WO 2015/048577 A2 April 2015 (02.04.2015) W P O P C T

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/048577 A2 April 2015 (02.04.2015) W P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 48/00 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/US20 14/057905 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 26 September 2014 (26.09.2014) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (25) Filing Language: English PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/883,925 27 September 2013 (27.09.2013) US (84) Designated States (unless otherwise indicated, for every 61/898,043 31 October 2013 (3 1. 10.2013) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: EDITAS MEDICINE, INC. [US/US]; 300 TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Third Street, First Floor, Cambridge, MA 02142 (US). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors: GLUCKSMANN, Alexandra; 33 Summit LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Road, Lexington, MA 02421 (US). PALESTRANT, De¬ SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, borah; 9 Avondale Road, Newton, MA 02459 (US). GW, KM, ML, MR, NE, SN, TD, TG). TARTAGLIA, Louis, Anthony; 23 Manor House Road, Newton, MA 02459 (US). MATA-FINK, Jordi; 8 Wind Published: sor Rd #1, Somerville, MA 02144 (US). CZECHOWICZ, — without international search report and to be republished Dorotz; 180 Commonwealth Ave, Unit B, Bo Agnieszka, upon receipt of that report (Rule 48.2(g)) ston, MA 021 16 (US). — with sequence listing part of description (Rule 5.2(a)) (74) Agent: BILLINGS, Nathan, A.; Lando & Anastasi LLP, Riverfront Office Park, One Main Street, Suite 1100, Cam bridge, MA 02142 (US).

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(54) Title: CRISPR-RELATED METHODS AND COMPOSITIONS (57) Abstract: Methods and compositions useful in targeting a payload to or editing a target nucleic acid are disclosed herein. CRISPR-RELATED METHODS AND COMPOSITIONS

FIELD OF THE INVENTION

The invention relates to CRISPR-related methods and components for editing of, or delivery of a payload to, a target nucleic acid sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 61/883,925, filed September 27, 2013; and U.S. Provisional Application No. 61/898,043, filed October 31, 2013. The contents of each of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 25, 2014, is named "C2159-7000WO_SL.txt" and is 210 KB in size.

BACKGROUND OF THE INVENTION

[0003] CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.

[0004] Recently, the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) allows for target sequence alteration through one of two endogenous DNA repair mechanisms —either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). The CRISPR/Cas system has also been used for gene regulation including transcription repression and activation without altering the target sequence. Targeted gene regulation based on the CRISPR/Cas system uses an enzymatically inactive Cas9 (also known as a catalytically dead Cas9).

SUMMARY OF THE INVENTION

[0005] Methods and compositions disclosed herein, e.g., a Cas9 molecule complexed with a gRNA molecule, can be used to target a specific location in a target DNA. Depending on the Cas9 molecule/gRNA molecule complex used specific editing or the delivery of a payload can be effected.

[0006] In one aspect, the disclosure features a gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25,

IX- 1, IX- 1A, IX-2, 1X-3, XIV- 1, or Section VIII.

[0007] In another aspect, the disclosure features a composition, e.g., pharmaceutical composition, comprising a gRNA molecule described herein.

[0008] In some embodiments, the composition further comprises a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule. In some embodiments, said Cas9 molecule is an eaCas9 molecule. In other embodiments, said Cas9 molecule is an eiCas9 molecule.

[0009] In some embodiments, said composition comprises a payload, e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.

[0010] In some embodiments, the payload comprises: an epigenetic modifier, e.g., a molecule that modifies DNA or chromatin; component, e.g., a molecule that modifies a histone, e.g., an epigenetic modifier described herein, e.g., in Section VI; a transcription factor, e.g., a transcription factor described herein, e.g., in Section VI; a transcriptional activator domain; an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitors; a small molecule; an antibody; an enzyme; an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase; and/or a nucleic acid, e.g., an enzymatically active nucleic acid, e.g., a ribozyme, or an mRNA, siRNA, of antisense oligonucleotide. In some embodiments, the composition further comprises a Cas9 molecule, e.g., an eiCas9, molecule. [0011] In some embodiments, said payload is coupled, e.g., covalently or noncovalently, to a Cas9 molecule, e.g., an eiCas9 molecule. In some embodiments, said payload is coupled to said Cas9 molecule by a linker. In some embodiments, said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions. In some embodiments, said linker is, or comprises, a bond described herein, e.g., in Section XI. In some embodiments, said linker is, or comprises, an ester bond. In some embodiments, said payload comprises a fusion partner fused to a Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule.

[0012] In some embodiments, said payload is coupled, e.g., covalently or noncovalently, to the gRNA molecule. In some embodiments, said payload is coupled to said gRNA molecule by a linker. In some embodiments, said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions. In some embodiments, said linker is, or comprises, a bond described herein, e.g., in Section XI. In some embodiments, said linker is, or comprises, an ester bond.

[0013] In some embodiments, the composition comprises an eaCas9 molecule. In some embodiments, the composition comprises an eaCas9 molecule which forms a double stranded break in the target nucleic acid.

[0014] In some embodiments, the composition comprises an eaCas9 molecule which forms a single stranded break in the target nucleic acid. In some embodiments, said single stranded break is formed in the complementary strand of the target nucleic acid. In some embodiments, said single stranded break is formed in the strand which is not the complementary strand of the target nucleic acid.

[0015] n some embodiments, the composition comprises HNH-like domain cleavage activity but having no, or no significant, N-terminal RuvC-like domain cleavage activity. In some embodiments, the composition comprises N-terminal RuvC-like domain cleavage activity but having no, or no significant, HNH-like domain cleavage activity.

[0016] In some embodiments, said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In some embodiments, said single stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. [0017] In some embodiments, the composition further comprises a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0018] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII-15, V1I-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV-1, or Section VIII.

[0019] In some embodiments, the template nucleic acid is or comprises a fragment of 0 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX- 3, XIV-1, or Section VIII.

[0020] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

0 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII- 15, VII-16, VII-17, VII-1 8, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV- 1, or Section VIII.

[0021] In some embodiments, the composition further comprises a second gRNA molecule, e.g., a second gRNA molecule described herein.

[0022] In some embodiments, said gRNA molecule and said second gRNA molecule mediate breaks at different sites in the target nucleic acid, e.g., flanking a target position. In some embodiments, said gRNA molecule and said second gRNA molecule are complementary to the same strand of the target. In some embodiments, said gRNA molecule and said second gRNA molecule are complementary to the different strands of the target.

[0023] In some embodiments, said Cas9 molecule mediates a double stranded break.

[0024] In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that first and second break made by the Cas9 molecule flank a target position. In some embodiments, said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0025] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0026] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of a target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VI1B, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17,

VII-18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV- 1, or Section VIII.

[0027] In some embodiments, the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to'200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g.', in Section V11B, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17,

VII-1 8, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV- 1, or Section VIII.

[0028] In some embodiments, the template nucleic acid is a fragment of 10 to 500, 10 to 400, 0 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1 , IX-IA, IX-2, IX- 3, XIV-1, or Section VIII.

[0029] In some embodiments, said Cas9 molecule mediates a single stranded break.

[0030] In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in the same strand of the nucleic acid target, e.g., in the case of transcribed sequence, the template strand or the non-template strand.

[0031] In some embodiments, said first and second break flank a target position.

[0032] In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. [0033] n some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position. In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII-

15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, ΓΧ - 1Α , rX-2, IX-3, XIV- 1, or Section VIII.

[0034] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 0 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-

3, XIV- , or Section VIII.

[0035] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

15, VII-16, VII-17, VII-1 8, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-1 A, IX-2, IX-3, XTV- 1, or Section VIII.

[0036] In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that a first and a second breaks are formed in different strands of the target. In some embodiments, said first and second break flank a target position. In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0037] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0038] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VIl-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, LX-3, XIV-1, o Section VIII.

[0039] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

0 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, X-2, IX- 3, XIV-1, or Section VIII.

[0040] n some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30 of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, LX-1A, IX-2, lX-3, XIV-1, or Section VIII.

[0041] In some embodiments, the composition comprises a second Cas9 molecule.

[0042] In some embodiments, one or both of said Cas9 molecule and said second Cas9 molecule are eiCas9 molecules. In some embodiments, said eiCas9 molecule is coupled to a payload by a linker and said second eiCas9 molecules is coupled to a second payload by a second linker.

[0043] In some embodiments, said payload and said second payload are the same. In some embodiments, said payload and said second payload are different. In some embodiments, said linker and said second linker are the same. In some embodiments, said linker and said second linker are different, e.g., have different release properties, e.g., different release rates.

[0044] In some embodiments, said payload and said second payload are each described herein, e.g., in Section VI, e.g., in Table VI- 1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7. In some embodiments, said payload and said second payload can interact, e.g., they are subunits of a protein.

[0045] In some embodiments, one of both of said Cas9 molecule and said second Cas9 molecule are eaCas9 molecules.

[0046] In some embodiments, said eaCas9 molecule comprises a first cleavage activity and said second eaCas9 molecule comprises a second cleavage activity. In some embodiments, said cleavage activity and said second cleavage activity are the same, e.g., both are N-terminal RuvC- like domain activity or are both HNH-like domain activity. In some embodiments, said cleavage activity and said second cleavage activity are different, e.g., one is N-terminal RuvC-like domain activity and one is HNH-like domain activity.

[0047] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for, e.g., NGGNG, NNAGAAW (W = A or T), or NAAR (R = A or G).

[0048] In some embodiments, said Cas9 molecule and said second Cas9 molecule both mediate double stranded breaks.

[0049] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that first and second break flank a target position. In some embodiments, one of said first and second double stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0050] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0051] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV-1, or Section VIII.

[0052] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX- 3, XIV-1, or Section VIII. [0053] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, rX-2, IX-3, XIV- , or Section VIII.

[0054] In some embodiments, one of said Cas9 molecule and said second Cas9 molecule mediates a double stranded break and the other mediates a single stranded break.

[0055] n some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, said first and second break flank a target position. In some embodiments, one of said first and second breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0056] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0057] n some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX- 3, XIV- 1, or Section VIII.

[0058] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII- 18, VII-19, VII-20, VII-21 , VTT-22, VII-23, VII-24, VII-25, IX-1 , IX- 1A, IX-2, IX- 3, XIV- 1, or Section VIII. [0059] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-1 A, IX-2, IX-3, XIV- 1, or Section VIII.

[0060] In some embodiments, said Cas9 molecule and said second Cas9 molecule both mediate single stranded breaks.

[0061] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said first and second break flank a target position.

[0062] In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0063] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments,- the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0064] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17,

VII- 18, VII- 19, ' VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV- 1, or Section VIII.

[0065] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-

XIV- 1, or Section VIII. [0066] n some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

15, VII-16, VII- 17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX-1A, IX-2, IX-3, XIV- 1, or Section VIII.

[0067] In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break are in the same strand.

[0068] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0069] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0070] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII-16, VII-17,

VII- 18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1A, IX-2, IX-3, XIV- 1, or Section VIII.

[0071] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16,

VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1A, IX-2, IX- 3, XIV- 1, or Section VIII.

[0072] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% 6f its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, Χ - 1Α,

IX-2, IX-3, XIV- 1, or Section VIII.

[0073] In some embodiments, said first and second break are on the different strands.

[0074] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another Pam described herein. In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break are on different strands.

[0075] In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, said first and second break flank a target position.

[0076] In some embodiments, one of said first and second single stranded breaks, or both are independently, within 0, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0077] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0078] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17, VII-1 8, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-1 A, IX-2, IX-3, XIV-1, or Section VIII.

[0079] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 0 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-1A, IX-2, IX- 3, XIV-1, or Section VIII. [0080] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

1 , VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1A,

IX-2, IX-3, XIV- 1, or Section VIII.

[0081] In yet another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a gRNA molecule and a second gRNA molecule described herein.

[0082] In some embodiments, the composition further comprises a nucleic acid, e.g., a DNA or mRNA, that encodes a Cas9 molecule described herein. In some embodiments, the composition further comprises a nucleic acid, e.g., a DNA or RNA, that encodes a second Cas9 molecule described herein. In some embodiments, the composition further comprises a template nucleic acid described herein.

[0083] In one aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes one or more gRNA molecules described herein.

[0084] In some embodiments, said nucleic acid comprises a promoter operably linked to the sequence that encodes a gRNA molecule, e.g., a promoter described herein.

[0085] In some embodiments, said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second gRNA molecule, e.g., a promoter described herein. In some embodiments, the promoter and second promoter are different promoters. In some embodiments, the promoter and second promoter are the same.

[0086] In some embodiments, the nucleic acid further encodes a Cas9 molecule described herein. In some embodiments, the nucleic acid further encodes a second Cas9 molecule described herein.

[0087] In some embodiments, said nucleic acid comprises a promoter operably linked to the sequence that encodes a Cas9 molecule, e.g., a promoter described herein.

[0088] In some embodiments, said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second Cas9 molecule, e.g., a promoter described herein. In some embodiments, the promoter and second promoter are different promoters. In some embodiments, the promoter and second promoter are the same.

[0089] In some embodiments, the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.

[0090] In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of: a) a Cas9 molecule, b) a second Cas9 molecule, c) a gRNA molecule, and d) a second gRNA molecule.

[0091] In some embodiments, each of a), b) c) and d) present are encoded on the same duplex molecule.

[0092] In some embodiments, a first sequence selected from of a), b), c) and d) is encoded on a first duplex molecule and a second sequence selected from a), b), c), and d) is encoded on a second duplex molecule.

[0093] In some embodiments, said nucleic acid encodes: a) and c); a), c), and d); or a), b), c), and d).

[0094] In some embodiments, the composition further comprises a Cas9 molecule, e.g., comprising one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.

[0095] In some embodiments, the composition further comprises an mRNA encoding Cas9 molecule, e.g., comprising one or more mRNAs encoding one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.

[0096] In some embodiments, the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.

[0097] In yet another aspect, the disclosure features a nucleic acid described herein.

[0098] n one aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV. [0099] In another aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

[01 00] n yet another aspect, the disclosure features a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

[0101] In still another aspect, the disclosure features a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g. , in Section IV.

[0102] In one aspect, the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with:

1) a composition comprising:

a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);

b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;

2) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; 3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; or 4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g.. in Section IV. [0103] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by, one dosage form, mode of delivery, or formulation.

[0104] In some embodiments, a) a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by, a first dosage form, a first mode of delivery, or a first formulation; and b) an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.

[0105] In some embodiments, the cell is an animal or plant cell. In some embodiments, the cell is a mammalian, primate, or human cell. In some embodiments, the cell is a human cell, e.g., a cell from described herein, e.g., in Section VIIA. In some embodiments, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In some embodiments, the cell is a human cell, e.g., a cancer cell or other cell characterized by a disease or disorder.

[0106] In some embodiments, the target nucleic acid is a chromosomal nucleic acid. In some embodiments, the target nucleic acid is an organellar nucleic acid. In some embodiments, the target nucleic acid is a mitochondrial nucleic acid. In some embodiments, the target nucleic acid is a chloroplast nucleic acid.

[0107] In some embodiments, the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

[0108] In some embodiments, the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

[0109] In some embodiments, said method comprises: modulating the expression of a gene or inactivating a disease organism.

[0110] In some embodiments, said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In some embodiments, said cell is a cell characterized by an unwanted genomic component, e.g., a viral genomic component. In some embodiments, the cell is a cell described herein, e.g., in Section ΠΑ . In some embodiments, a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.

[0111] In some embodiments, the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.

[0112] In some embodiments, the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In some embodiments, said composition comprises a template nucleic acid.

[0113] In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[01 14] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, V1I-20, VII, 21, VIl-22, Vl -23, VII-24, VII-25, IX-1, IX-IA, 1X-2, IX-3, XIV-

1, or Section VIII.

[01 15] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VlI-17, Vll-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-

3, XIV- 1, or Section VIII.

[0116] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a con-esponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII- 15, VII-16, VII-17, VII-1 8, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA,

IX-2, IX-3, XIV- 1, or Section VIII.

[0 17] In some embodiments, a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted; c) the coding sequence of a gene is cleaved; d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected; and/or e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.

[0118] In some embodiments, a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered. [01 ] n another aspect, the disclosure features a method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of:

1) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ; b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; 2) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; 3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and/or 4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

[0120] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.

[0121] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, in a first mode of delivery, or first formulation; and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.

[0122] n some embodiments, the subject is an animal or plant. In some embodiments, the subject is a mammalian, primate, or human.

[0123] In some embodiments, the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section V A. In some embodiments, the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.

[0124] In some embodiments, the target nucleic acid is a chromosomal nucleic acid. In some embodiments, the target nucleic acid is an organellar nucleic acid. In some embodiments, the nucleic acid is a mitochondrial nucleic acid. In some embodiments, the nucleic acid is a chloroplast nucleic acid.

[0125] In some embodiments, the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite. In some embodiments, said method comprises modulating expression of a gene or inactivating a disease organism.

[0126] In some embodiments, the target nucleic acid is the nucleic acid of a cell characterized by unwanted proliferation, e.g., a cancer cell. In some embodiments, said target nucleic acid comprises an unwanted genomic component, e.g., a viral genomic component. In some embodiments, a control or structural sequence of at least, 2 3, 4, or 5 genes is altered. In some embodiments, the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a rumor suppressor.

[0127] In some embodiments, the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

[0128] In some embodiments, said composition comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

[0129] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17,

VII-18. VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX-1 A, IX-2, IX-3, XIV-1, or Section V l.

[0130] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 0 to 300, 0 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table Vll-13, VII-14, VII-15, VI1-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-

3, XIV- 1, or Section VIII.

[0131] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500,

10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:

[0132] In some embodiments,

a) a control region, e.g., a cis-acting or trans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or trans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted; c) the coding sequence of a gene is cleaved; d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected; e) the non-coding sequence of a gene or an intergenic region between genes is cleaved; and/or

f) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.

[0133] In some embodiments, a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.

[0134] In one aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non- covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule.

[0135] In another aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule.

[0136] In yet another aspect, the disclosure features a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or noh-covalently, to the Cas9 molecule.

[0137] In still another aspect, the disclosure features a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.

[0138] n one aspect, the disclosure features a method of delivering a payload to a cell, e.g., by targeting a payload to target nucleic acid, comprising contacting said cell with:

1) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule; 2) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule; 3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and/or 4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA .encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.

[0139] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.

[0140] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, first mode of delivery, or first formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.

[0141] In some embodiments, the cell is an animal or plant cell. In some embodiments, the cell is a mammalian, primate, or human cell. In some embodiments, the cell is a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In some embodiments, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In some embodiments, the cell is a human cell, e.g., a cancer cell, a cell comprising an unwanted genetic element, e.g., all or part of a viral genome.

[0142] In some embodiments, the gRNA mediates targeting of a chromosomal nucleic acid. In some embodiments, the gRNA mediates targeting of a selected genomic signature. In some embodiments, the gRNA mediates targeting of an organellar nucleic acid. In some embodiments, the gRNA mediates targeting of a mitochondrial nucleic acid. In some embodiments, the gRNA mediates targeting of a chloroplast nucleic acid.

[0143] In some embodiments, the cell is a cell of a disease causing organism, e.g., a vims, bacterium, fungus, protozoan, or parasite. [0144] In some embodiments, the gR A mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

[0145] In some embodiments, the payload comprises a payload described herein, e.g., in Section VI.

[0146] In some embodiments, said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In some embodiments, said cell is characterized by an unwanted genomic component, e.g., a viral genomic component.

[0147] In some embodiments, a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.

[0148] In some embodiments, the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation. In some embodiments, the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or tumor suppressor. In some embodiments, the gRNA targets a cancer cell, e.g., a cancer cell disclosed herein, e.g., in Section V A. In some embodiments, the gRNA targets a cell which has been infected with a virus.

[0149] In another aspect, the disclosure features a method of treating a subject, e.g., by targeting a payload to target nucleic acid, comprising administering to the subject, an effective amount of:

1) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule; 2) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or is a fusion partner with the Cas9 molecule; 3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and/or 4) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.

[0150] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.

[0151] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage, mode of delivery form or formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, mode of delivery, or formulation.

[0152] In some embodiments, the subject is an animal or plant cell. In some embodiments, the subject is a mammalian primate, or human cell. [0153] In some embodiments, the gRNA mediates targeting of a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In some embodiments, the gRNA mediates targeting of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In some embodiments, the gRNA mediates targeting of a cancer cell or a cell comprising an unwanted genomic element, e.g., all or part of a viral genome. In some embodiments, the gRNA mediates targeting of a chromosomal nucleic acid. In some embodiments, the gRNA mediates targeting of a selected genomic signature. In some embodiments, the gRNA mediates targeting of an organellar nucleic acid. In some embodiments, the gRNA mediates targeting of a mitochondrial nucleic acid. In some embodiments, the gRNA mediates targeting of a chloroplast nucleic acid. In some embodiments, the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite. In some embodiments, the gRNA targets a cell characterized by unwanted proliferation, e.g., a cancer cell, e.g., a cancer cell from Section VIIA, e.g., from Table VII- 11. In some embodiments, the gRNA targets a cell characterized by an unwanted genomic component, e.g., a viral genomic component.

[0154] In some embodiments, a control element, e.g., a promoter or enhancer, is targeted. In some embodiments, the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor. In some embodiments, the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.

[0155] In some embodiments, the gRNA targets a cancer cell. In some embodiments, the gRNA targets a cell which has been infected with a virus.

[0156] In some embodiments, at least one eaCas9 molecule and a payload are administered. In some embodiments, the payload comprises a payload described herein, e.g., in Section VI.

[0157] In one aspect, the disclosure features a reaction mixture comprising a composition described herein and a cell.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0 159] Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.

[01 0] Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.

BRIEF DESCRIITION OF THE DRAWING

[0161] The Figures described below, that together make up the Drawing, are for illustration purposes only, not for limitation.

[0 162] FIG. 1A-G are representations of several exemplary gRNAs.

[01 63] FIG. A depicts a modular gRNA molecule derived in part (or modeled on a sequence in part) from Streptococcus pyogenes {S. pyogenes) as a duplexed structure (SEQ ID NOS 42 and 43, respectively, in order of appearance);

[01 64] FIG. B depicts a unimolecular (or chimeric) gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 44);

[01 65] FIG. C depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45);

[01 66] FIG. D depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 46);

[01 67] FIG. E depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 47);

[01 68] FIG. I F depicts a modular gRNA molecule derived in part from Streptococcus

Ihermophilus (S. Ihermophilus) as a duplexed structure (SEQ ID NOS 48 and 49, respectively, in order of appearance);

[01 69] FIG. 1G depicts an alignment of modular gRNA molecules of S. pyogenes and S. Ihermophilus (SEQ ID NOS 50-53, respectively, in order of appearance). [0170] FIG. 2 depicts an alignment of Cas9 sequences from Chylinski et al., RNA BIOL. 2013; 10(5): 726-737. The N-terminal RuvC-like domain is boxed and indicated with a "Y". The other two RuvC-like domains are boxed and indicated with a "B". The HNH-like domain is boxed and indicated by a "G". Sm: S. mutatis (SEQ ID NO: 1); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophilus (SEQ ID NO: 3); Li: L. innocua (SEQ ID NO: 4). Motif: this is a motif based on the four sequences: residues conserved in all four sequences are indicated by single letter amino acid abbreviation; "*" indicates any amino acid found in the corresponding position of any of the four sequences; and "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids.

[0171] FIG. 3A shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 54-103, respectively, in order of appearance). The last line of FIG. 3A identifies 3 highly conserved residues.

[0172] FIG. 3B shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 104-177, respectively, in order of appearance). The last line of FIG. 3B identifies 4 highly conserved residues.

[0173] FIG. 4A shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 178-252, respectively, in order of appearance). The last line of FIG. 4A identifies conserved residues.

[01 74] FIG. 4B shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 253-302, respectively, in order of appearance). The last line of FIG. 4B identifies 3 highly conserved residues.

[01 75] FIG. 5 depicts an alignment of Cas9 sequences from S. pyogenes and Neisseria meningitidis (N. meningitidis). The N-terminal RuvC-like domain is boxed and indicated with a "Y". The other two RuvC-like domains are boxed and indicated with a "B". The HNH-like domain is boxed and indicated with a "G". Sp: S. pyogenes; Nm: N. meningitidis. Motif: this is a motif based on the two sequences: residues conserved in both sequences are indicated by a single amino acid designation; "*" indicates any amino acid found in the corresponding position of any of the two sequences; "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, and "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.

[01 76] FIG. 6 shows a nucleic acid sequence encoding Cas9 of N. meningitidis (SEQ ID NO: 303). Sequence indicated by an "R" is an SV40 NLS; sequence indicated as "G" is an HA tag; sequence indicated by an "O" is a synthetic NLS sequence. The remaining (unmarked) sequence is the open reading frame (ORF).

DEFINITIONS

[01 77] "Domain", as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.

[0 178] Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, in some embodiments, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

[01 79] "Modulator", as used herein, refers to an entity, e.g., a drug, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence. In an embodiment, modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non- covalent bond, e.g., the attachment of a moiety, to the subject molecule. In an embodiment, a modulator alters the, three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule. A modulator can increase, decrease, initiate, or eliminate a subject activity. [0180] "Large molecule", as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologies, and carbohydrates. [0181] "Polypeptide", as used herein, refers to a polymer of amino acids having less than 100 amino acid residues. In an embodiment, it has less than 50, 20, or 10 amino acid residues. [0182] "Reference molecule", e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made. [0183] "Replacement", or "replaced", as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present. [0184] "Small molecule", as used herein, refers to a compound having a molecular weight less than about 2 kD, e.g., less than about 2 kD, less than about 1.5 kD, less than about 1 kD, or less than about 0.75 kD. [0185] "Subject", as used herein, may mean either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In an embodiment, the subject is a human. In other embodiments, the subject is poultry. [0186] "Treat", "treating" and "treatment", as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; or (c) curing the disease. [0187] "X" as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.

DETAILED DESCRIPTION

I. gRNA Molecules [0188] A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid. gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as "chimeric" gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below.

[0189] Several exemplary gRNA structures, with domains indicated thereon, are provided in FIG. 1. While not wishing to be bound by theory with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in FIG. 1 and other depictions provided herein.

[0190] In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5 to 3': a targeting domain (which is complementary to a target nucleic acid); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain. In an embodiment, a modular gRNA comprises: a first strand comprising, preferably from 5' to 3'; a targeting domain (which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII- 20, V -2 , VII-22, VI1-23, VII-24, VII-25, IX- 1, IX- A, LX-2, 1X-3, XlV-1, or Section VIII); and a first complementarity domain; and a second strand, comprising, preferably from 5' to 3': optionally, a 5' extension domain; a second complementarity domain; and a proximal domain; and optionally, a tail domain.

[0191] The domains are discussed briefly below:

1) The Targeting Domain:

[0192] FIG. 1A-G provides examples of the placement of targeting domains.

[0193] The targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises, in the 5' to 3' direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence.

In an embodiment, the targeting domain is 5 to 50, e.g., 10 to 40, e.g., 10 to 30, e.g., 15 to 30, e.g., 1 to 25 nucleotides in length. In an embodiment, the targeting domain is 15, 16, 17, 18,

19, 20, 2 1, 22, 23, 24 or 25 nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.

[0194] In an embodiment, the targeting domain is 16 nucleotides in length. [0195] In an embodiment, the targeting domain is 17 nucleotides in length.

[0196] In an embodiment, the targeting domain is 18 nucleotides in length.

[0197] In an embodiment, the targeting domain is 1 nucleotides in length.

[0198] In an embodiment, the targeting domain is 20 nucleotides in length.

[0199] In an embodiment, the targeting domain is 2 nucleotides in length.

[0200] In an embodiment, the targeting domain is 22 nucleotides in length.

[0201] In an embodiment, the targeting domain is 23 nucleotides in length.

[0202] In an embodiment, the targeting domain is 24 nucleotides in length.

[0203] In an embodiment, the targeting domain is 25 nucleotides in length.

[0204] Targeting domains are discussed in more detail below.

2) The First Complementarity Domain :

[0205] FIG. lA-G provides examples of first complementarity domains.

[0206] The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an · embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 22 nucleotides in length. In an embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an embodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1, 22, 23, 24, or 25 nucleotides in length.

[0207] In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is I, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3' subdomain is 3 to 25, e.g., 4-22, 4-18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 5, 16, 7, 18, 19, 20, 2 1, 22, 23, 24, or 25, nucleotides length.

[0208] The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thennophilus, first complementarity domain.

[0209] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.

[0210] First complementarity domains are discussed in more detail below.

3) The Linking Domain

[0211] FIG. 1B-E provides examples of linking domains.

[0212] A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains, see, e.g., FIG. 1B-E. In an embodiment, the linking domain is, or comprises, a covalent bond inteiposed between the first complementarity domain and the second complementarity domain. Typically, the linking domain comprises one or more, e.g., 2, 3, 4, 5,

6, 7, 8, 9, or 10 nucleotides.

[0213] In modular gRNA molecules the two molecules can be associated by virtue of the hybridization of the complementarity domains, see e.g., FIG. 1A.

[0214] A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length.

[0215] In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5' to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.

[0216] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.

[0217] Linking domains are discussed in more detail below.

4) The 5 Extension Domain

[0218] In an embodiment, a modular gRNA can comprise additional sequence, 5 to the second complementarity domain, referred to herein as the 5' extension domain, see, e.g., FIG. 1A. In an embodiment, the 5' extension domain is, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 nucleotides in length. In an embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

5) The Second Complementarity Domain :

[021 ] FIG. A-F provides examples of second complementarity domains.

[0220] The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, e.g., as shown in FIG. 1A or FIG. IB, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.

[0221] In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length.

In an embodiment, it is longer than the first complementarity region.

[0222] In an embodiment, the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24 or 25 nucleotides in length. [0223] In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 tol8, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.

[0224] In an embodiment, the 5' subdomain and the 3' subdomain of the first complementarity domain, are respectively, complementaiy, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.

[0225] The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, first complementarity domain.

[0226] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.

6) A Proximal Domain :

[0227] FIG. 1A-F provides examples of proximal domains.

[0228] In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, proximal domain.

[0229] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.

7) A Tail Domain :

[0230] FIG. A and FIG. 1C-F provide examples of tail domains.

[0231] As can be seen by inspection of the tail domains in FIG. 1A and FIG. 1C-F, a broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, the tail domain nucleotides a e from or share homology with sequence from the 5 end of a naturally occurring tail domain, see e.g., FIG. D or FIG. IE. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.

[0232] In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, tail domain.

[0233] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.

[0234] In an embodiment, the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3' end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When altemate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.

[0235] The domains of gRNA molecules are described in more detail below.

The Targeting Domain

[0236] The "targeting domain" of the gRNA is complementary to the "target domain" on the target nucleic acid. The strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of th e gRNA is referred to herein as the "complementary strand" of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y el a!.. NAT BIOTECHNOL 2014 (doi: 10.1038/nbt.2808) and Sternberg SH el a/..

NATURE 2014 (doi: 10. 1038/naturel301 1).

[0237] In an embodiment, the targeting domain is 16, 17, 18, , 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0238] In an embodiment, the targeting domain comprises 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0239] In an embodiment, the targeting domain is 16 nucleotides in length. [0240] In an embodiment, the targeting domain is 17 nucleotides in length.

[0241] In an embodiment, the targeting domain is 18 nucleotides in length.

[0242] In an embodiment, the targeting domain is 9 nucleotides in length.

[0243] In an embodiment, the targeting domain is 20 nucleotides in length.

[0244] In an embodiment, the targeting domain is 2 1 nucleotides in length.

[0245] In an embodiment, the targeting domain is 22 nucleotides in length.

[0246] In an embodiment, the targeting domain is 23 nucleotides in length.

[0247] In an embodiment, the targeting domain is 24 nucleotides in length.

[0248] In an embodiment, the targeting domain is 25 nucleotides in length.

[0249] In an embodiment, the targeting domain is 10 +/-5, 20+/-5, 30+/-5, 40+/- 5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.

[0250] In an embodiment, the targeting domain is 20+/-5 nucleotides in length.

[0251] In an embodiment, the targeting domain is 20+/-10, 30+/-10, 40+/-10, 50+/-10, 60+/-10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.

[0252] In an embodiment, the targeting domain is 30+/- 10 nucleotides in length.

[0253] In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other embodiments, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

[0254] Typically the targeting domain has full complementarity with the target sequence. In some embodiments the targeting domain has or includes 1, 2, 3, 4, .5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.

[0255] In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end. [0256] In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end.

[0257] In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

[0258] In some embodiments, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain ("non-complementary nucleotides"), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

[0259] In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.

[0260] In an embodiment, there are no noncomplementary nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

[0261] In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the targeting domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' acetylation, e.g., a 2' methylation, or other modification from Section X . [0262] In some embodiments, the targeting domain includes 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the targeting domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.

[0263] In some embodiments, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5 end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

[0264] In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

[0265] Modifications in the targeting domain can be selected so as to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ΠΙ. gRNA's having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section III. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

[0266] In some embodiments, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In other embodiments, 1, 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.

[0267] In an embodiment, the targeting domain comprises, preferably in the 5' → 3' direction: a secondary domain and a core domain. These domains are discussed in more detail below. The Core Domain and Secondary Domain of the Targeting Domain

[0268] The "core domain" of the targeting domain is complementary to the "core domain target" on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain).

[0269] In an embodiment, the core domain is 6 +/-2, 1+1-2, 8+/-2, 9+/-2, 10+/-2, 1+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, or 16+-2 nucleotides in length.

[0270] In an embodiment, the core domain is 10+/-2 nucleotides in length.

[0271] In an embodiment, the core domain is 10+/-4 nucleotides in length.

[0272] In an embodiment, the core domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides in length.

[0273] In an embodiment, the core domain is 8 to 13, e.g., 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to , 9 to 1 , 9 to 1, or 9 to 0 nucleotides in length.

[0274] In an embodiment, the core domain is 6 to 16, e.g., 6 to 15, 6 to 14, 6 to 13, 7 to 14, 7 to

13, 7 to 12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 2, 8 to 11, 8 to 10, or 8 to 9 nucleotides in length.

[0275] The core domain is complementary with the core domain target. Typically the core domain has exact complementarity with the core domain target. In some embodiments, the core domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

[0276] The "secondary domain" of the targeting domain of the gRNA is complementary to the "secondary domain target" of the target nucleic acid.

[0277] In an embodiment, the secondary domain is positioned 5' to the core domain.

[0278] In an embodiment, the secondary domain is absent or optional. [0279] n an embodiment, if the targeting domain is 25 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 2 to 17 nucleotides in length.

[0280] In an embodiment, if the targeting domain is 24 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 11 to 16 nucleotides in length.

[0281] In an embodiment, if the targeting domain is 23 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 5 nucleotides in length.

[0282] In an embodiment, if the targeting domain is 22 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.

[0283] In an embodiment, if the targeting domain is 2 1 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.

[0284] In an embodiment, if the targeting domain is 20 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.

[0285] In an embodiment, if the targeting domain is 19 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 11 nucleotides in length.

[0286] In an embodiment, if the targeting domain is 18 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.

[0287] In an embodiment, if the targeting domain is 17 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length. [0288] n an embodiment, if the targeting domain is 16 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.

[0289] In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or

15 nucleotides in length.

[0290] The secondary domain is complementary with the secondary domain target. Typically the secondary domain has exact complementarity with the secondary domain target. In some embodiments the secondary domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the secondary domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

[0291] In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification from Section X . In an embodiment, a nucleotide of the core domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X. Typically, a core domain will contain no more than 1, 2, or 3 modifications.

[0292] Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section

Π1. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.

[0293] In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification from Section X . In an embodiment, a nucleotide of the secondary domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X. Typically, a secondary domain will contain no more than 1, 2, or 3 modifications.

[0294] Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.

[0295] In an embodiment, (1) the degree of complementarity between the core domain and its target, and (2) the degree of complementarity between the secondary domain and its target, may differ. In an embodiment, (1) may be greater than (2). In an embodiment, (1) may be less than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be completely complementary with its target.

[0296] In an embodiment, (1) the number of modifications (e.g., modifications from Section X) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section X) of the nucleotides of the secondaiy domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be free of modifications.

The First and Second Complementarity Domains

[0297] The first complementarity domain is complementary with the second complementarity domain.

[0298] Typically the first domain does not have exact complementarity with the second complementarity domain target. In some embodiments, the first complementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain . In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1, 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5' end of the second complementarity domain.

[0299] In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

[0300] In an embodiment, the first and second complementarity domains are:

independently, 6 +1-2, 7+/-2, 8+/-2, 9+/-2, 10+/-2, 11+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, 16+/-2, 17+/-2, 18+/-2, 19+/-2, or 20+/-2, 21+/-2, 22+A2, 23+ 2, or 24+/-2 nucleotides in length;

independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length; or independently, 5 to 24, 5 to 23, 5 to 22, 5 to 2 1, 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.

[0301 ] In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.

[0302] In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section X.

[0303] In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification from Section X . In an embodiment, a nucleotide of the domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g. , a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.

[0304] In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the first and second complementary domains, independently, include as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.

[0305] In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3 end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.

[0306] Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section III. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.

[0307] In an embodiment, the first complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. lliermophilus, first complementarity domain, or a first complementarity domain described herein, e.g., from FIG. 1A-F.

[0308] In an embodiment, the second complementarity domain has at least 60, 70, 80, 85%,

90%, or 95 % homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermopliilus, second complementarity domain, or a second complementarity domain described herein, e.g., from FIG. A-F.

[0309] The duplexed region formed by first and second complementarity domains is typically 6,

7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).

[0310] In some embodiments, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNN GUUUUAG AGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 5).

[0311] In some embodiments, the first and second complementarity domains, when duplexed, comprise 1 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNN GUUUUAG AGCUAUGCU GAAAAGC AUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGC (SEQ ID NO: 27).

[0312] In some embodiments the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNN GUUUUAG AGCUAUGCUG GAAACA GCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGC (SEQ ID NO: 28).

[0313] In some embodiments the first and second complementarity domains, when duplexed, comprise 2 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGG AAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 29). [0314] n some embodiments, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):

NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAG UUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 30);

NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAG UUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 31); and

NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGG AAACAAUACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 32).

The 5' Extension Domain

[0315] In an embodiment, a modular gRNA can comprise additional sequence, 5' to the second complementarity domain. In an embodiment, the 5' extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

[0316] In an embodiment, the 5' extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X . However, in an embodiment, the 5' extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5 extension domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the 5' extension domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' - acetylation, e.g., a 2' methylation, or other modification from Section X.

[0317] In some embodiments, the 5' extension domain can comprise as many as 1, 2, 3, 4, 5, 6, 7

or 8 modifications. In an embodiment, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule. [0318] I some embodiments, the 5' extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5 ' end of the 5' extension domain, within 5 nucleotides of the 3 ' end of the 5 ' extension domain, or more than 5 nucleotides away from one or both ends of the 5 ' extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the 5 extension domain, within 5 nucleotides of the 3 ' end of the 5 ' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5 ' extension domain.

n an embodiment, no nucleotide is modified within 5 nucleotides of the 5 ' end of the 5 '

extension domain, within 5 nucleotides of the 3 ' end of the 5 ' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5 ' extension domain.

[0319] Modifications in the 5 ' extension domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system

described in Section ITT. gRNAs having a candidate 5 ' extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate 5 ' extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

[0320] In an embodiment, the 5 ' extension domain has at least 60, 70, 80, 85, 90 or 95%

homology with, or differs by no more than 1, 2, 3, 4 , 5, or 6 nucleotides from, a reference 5 ' extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, 5' extension domain, or a 5 ' extension domain described herein, e.g., from FIG. 1A and FIG. IF.

The Linking Domain

[0321] In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.

[0322] In an embodiment, the linking domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.

[0323] In an embodiment, the linking domain is 20+/-10, 30+/-10, 40+/-10, 50+/- 10, 60+/-10, 70+/-10, 80+/-10, 90+/-10, or 100+/- 10 nucleotides, in length. [0324] In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60,

10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other embodiments, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

[0325] In an embodiment, the linking domain is 1, 2, 3, 4, 5,.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.

[0326] In an embodiment, the linking domain is a covalent bond.

[0327] In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1, 2, or 3 nucleotides of the 3 end of the first complementarity domain and/or the fi end of the second complementarity domain. In an embodiment, the duplexed region can be 20+/- 10, 30+/- 10, 40, +/-10 or 50+/- 10 base pairs in length. In an embodiment, the duplexed region can be 10+/-5, 15+/-5, 20+/-5, or 30+/-5 base pairs in length. In an embodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length.

[0328] Typically the sequences forming the duplexed region have exact complementarity with one another, though in some embodiments as many as 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.

[0329] In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment the linking . domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other modification from Section X . In an embodiment, a nucleotide of the linking domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.

[0330] In some embodiments, the linking domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.

[0331] Modifications in a linking domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ΠΙ. gRNA's having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section III. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

[0332] In an embodiment, the linking domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5 ,or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein, e.g., from FIG. 1B-E.

The proximal domain

[0333] In an embodiment, the proximal domain is 6 +1-2, Ί +/-2, 8+/-2, 9+/-2, 10+/-2, 11+/-2, 12+/-2, 13+/-2, 14+/-2, 14+/-2, 16+/-2, 17+/-2, 18+/-2, 19+/-2, or 20+/-2 nucleotides in length.

[0334] n an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, or 20 nucleotides in length.

[0335] In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.

[0336] In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the proximal domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' - acetylation, e.g., a 2' methylation, or other modification from Section X.

[0337] In some embodiments, the proximal domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or

8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.

[0338] In some embodiments, the proximal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.

[0339] Modifications in the proximal domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated:

[0340] In an embodiment, the proximal domain has at least 60%, 70%, 80%, 85%, 90%, or 95% homology with, or differs by no more thari 1, 2, 3, 4, 5 ,or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilics, proximal domain, or a proximal domain described herein, e.g., from FIG. 1A-F.

The Tail Domain

[0341] n an embodiment, the tail domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 00+/- 5 nucleotides, in length.

[0342] In an embodiment, the tail domain is 20+/-5 nucleotides in length.

[0343] In an embodiment, the tail domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/-

10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.

[0344] In an embodiment, the tail domain is 25+/- 10 nucleotides in length.

[0345] In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 0 to 15 nucleotides in length. [0346] In other embodiments, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

[0347] In an embodiment, the tail domain is 1 to 20, 1 to 1, 1 to 10, or 1 to 5 nucleotides in length.

[0348] In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the tail domain can comprise a 2' modification (e.g., a modification at the position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.

[0349] n some embodiments, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.

[0350] In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In an embodiment, a further single stranded domain, exists 3' to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, it is 4 to 6 nucleotides in length.

[0351] In an embodiment, the tail domain has at least 60, 70, 80, or 90% homology with, or differs by no more than , 2, 3, 4, 5 ,or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, tail domain, or a tail domain described herein, e.g., from FIG. 1A and FIG. 1C-F.

[0352] In an embodiment, the proximal and tail domain, taken together comprise the following sequences: AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU (SEQ D NO: 33); AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GGUGC (SEQ ID NO: 34);

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCGGAUC (SEQ ID NO: 35);

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO: 36);

AAGGCUAGUCCGUUAUCA (SEQ ID NO: 37); or

AAGGCUAGUCCG (SEQ ID NO: 38).

[0353] In an embodiment, the tail domain comprises the 3' sequence UUUUUU, e.g., if a U6 promoter is used for transcription.

[0354] In an embodiment, the tail domain comprises the 3' sequence UUUU, e.g., if an H I promoter is used for transcription.

[0355] In an embodiment, tail domain comprises variable numbers of 3' U's depending, e.g., on the termination signal of the pol-III promoter used.

[0356] In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template if a T7 promoter is used.

[0357] In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.

[0358] In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template, e.g, if a pol-II promoter is used to drive transcription.

[0359] Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ΠΙ. gRNA's having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section ΠΙ. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

[0360] n some embodiments, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.

[0361] In an embodiment a gRNA has the following structure: [0362] 5' [targeting domain]-[first complementarity domain]-[linking domain]-[second complementarity domain]-[proximal domain]-[tail domain]-3' wherein, the targeting domain comprises a core domain and optionally a secondary domain, and is

10 to 50 nucleotides in length; the first complementarity domain is 5 to 25 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90, or 95% homology with a reference first complementarity domain disclosed herein; the linking domain is 1 to 5 nucleotides in length; the proximal domain is 5 to 20 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference proximal domain disclosed herein; and the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference tail domain disclosed herein.

Exemplary Chimeric gRNAs [0363] In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5' to 3': a targeting domain (which is complementary to a target nucleic acid); a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and a tail domain, wherein, (a) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 6, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain. [0364] In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

[0365] In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0366] In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0367] In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 5 1, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0368] In an embodiment, the targeting domain has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 2 1, 22, 23, 24 or 25 nucleotides in length.

[0369] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length. [0370] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 7 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 7 nucleotides in length.

[0371] n an embodiment, the targeting domain has, or consists of, 8 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 8 nucleotides in length.

[0372] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 1 nucleotides in length.

[0373] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

[0374] In an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 2 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length.

[0375] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

[0376] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

[0377] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

[0378] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.

[0379] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 6 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0380] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting . domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0381] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0382] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0383] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0384] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g.; 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0385] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together; comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. [0386] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0387] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0388] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0389] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0390] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0391] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0392] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0393] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0394] n an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 2 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 1 , 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0395] In an embodiment, the targeting domain has, or consists of, 2 nucleotides (e.g., 2 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0396] In an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 2 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0397] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0398] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain. [0399] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting

domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 5 1, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0400] n an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least , 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0401] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, ( or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0402] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0403] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0404] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0405] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3 to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain:

[0406] n an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0407] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3 to the last nucleotide of the second complementarity domain.

[0408] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

Exemplary Modular gRNAs [0409] In an embodiment, a modular gRNA comprises: a first strand comprising, preferably from 5' to 3'; a targeting domain; a first complementarity domain; and a second strand, comprising, preferably from 5' to 3': optionally a 5' extension domain; a second complementarity domain; a proximal domain; and a tail domain, wherein: (a) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 3 1, 35, 40, 45, 49, 50, or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3 to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0410] In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

[041 1] In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0412] In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0413] In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0414] In an embodiment, the targeting domain has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, , 18, 19, 20, 2 1, 22, 23, 24 or 25 nucleotides in length.

[0415] I n an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.

[0416] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

[0417] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 8 nucleotides in length.

[0418] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length. [0419] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

[0420] In an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 2 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length.

[0421] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

[0422] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

[0423] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

[0424] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.

[0425] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0426] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 6 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 3 1, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0427] n an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0428] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0429] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0430] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0431] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0432] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 5 1, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain. [0433] In an embodiment, the targeting domain has, or consists of, 1 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0434] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0435] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0436] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0437] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting

domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35-, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0438] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0439] In an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0440] In an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 2 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 2 1 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3 to the last nucleotide of the second complementarity domain.

[0441] In an embodiment, the targeting domain has, or consists of, 2 1 nucleotides (e.g., 2 1 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting

domain is 2 1 nucleotides in length; and there are at least 16, 19, 21, 26, 3 1, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0442] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0443] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0444] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 3 1, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0445] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. [0446] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0447] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0448] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0449] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0450] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

[0451] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 3 1, 35, 40, 45, 49, 50, or 53 nucleotides.

[0452] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0453] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

Methods for Designing gRNAs

[0454] Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein. Targeting Domains discussed herein can be incorporated into the gRNAs described herein.

[0455] Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in. ali e al., 2013 SCIENCE 339(6121): 823-826; Hsu et al, 2013 NAT

BIOTECHNOL, 3 1(9): 827-32; Fu et al, 2014 NAT BIOTECHNOL, doi: 10.1038/nbt.2808. PubMed PMID: 24463574; Heigwer et al, 2014 NAT METHODS l l(2):122-3. doi: 10.1038/nmeth.2812. PubMed PMID: 24481216; Bae e al, 2014 BIOINFORMATICS PubMed PMID: 24463181; Xiao A el al, 2014 BIOINFORMATICS PubMed PMID: 24389662.

[0456] For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice e.g., using S. pyogenes Cas9, the tool can identify all off-target sequences (e.g., preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for CR1SPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein. II. Cas9 Molecules

[0457] Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description

herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitidis Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhiz' obium sp., Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacler diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, llyobacler polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica. Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

[0458] A Cas9 molecule, as that term is used herein, refers to a molecule that can interact with a gRNA molecule and, in concert with the gRNA molecule, localize (e.g., target or home) to a site which comprises a target domain and PAM sequence. .

[0459] In an embodiment, the Cas9 molecule is capable of cleaving a target nucleic acid molecule. A Cas9 molecule that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule. In an embodiment, an eaCas9 molecule, comprises one or more of the following activities:

[0460] a nickase activity, i.e.,.the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities; an endonuclease activity; an exonuclease activity; and a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.

[0461] In an embodiment, an enzymatically active Cas9 or an eaCas9 molecule cleaves both DNA strands and results in a double stranded break. In an embodiment, an eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH-like domain and an active, or cleavage competent, N-terminal RuvC-like domain.

[0462] In an embodiment, the ability of an eaCas9 molecule to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to

5, base pairs upstream from that sequence. See, e.g., Mali el ai, SCIENCE 2013; 339(6121): 823- 826. In an embodiment, an eaCas9 molecule of S. tliermophilus recognizes the sequence motif NGGNG and NNAG AAW (W = A or T) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al., SCIENCE 2010; 327(5962): 167-170, and Deveau et al, J BACTERIOL 2008; 190(4): 1390- 1400. In an embodiment, an eaCas9 molecule of S. mulans recognizes the sequence motif NGG or NAAR (R - A or G) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R = A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g.,

Hou et al., PNAS EARLY EDITION 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al, SCIENCE 2012, 337:816.

[0463] Some Cas9 molecules have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule home (e.g., targeted or localized) to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 (an enzymatically inactive Cas9) molecule. For example, an eiCas9 molecule can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1 % of the cleavage activity of a reference Cas9 molecule, as measured by an assay described herein.

[0464] Exemplary naturally occurring Cas9 molecules are described in Chylinski et al, RNA Biology 2013; 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 0 bacterial family, a cluster 1 bacterial family, a cluster 12 bacterial family, a cluster 3 bacterial family, a cluster 14 bacterial family, a cluster 1 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 8 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 2 1 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 3 1 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 4 1 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family,. a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 1 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 6 1 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 7 1 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.

[0465] Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI- 1), S. tliermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. /nutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC1 1558), S. gallolylicus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. cmginosus

(e.g.; strain F021 1), S. agalactia* (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clipl 1262), EtUerococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et al. PNAS Early Edition 2013, 1-6) and a S. aureus Cas9 molecule.

[0466] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with; differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared with;

differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or is identical to; any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA Biology

2013, 10:5, Ί 2Ί -Τ ,1 Hou et al. PNAS Early Edition 2013, 1-6. In an embodiment, the Cas9 molecule comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to localize to a target nucleic acid.

[0467] In an embodiment, a Cas9 molecule comprises the amino acid sequence of the consensus sequence of FIG. 2, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutatis and L. innocua, and "-" indicates any amino acid. In an embodiment, a Cas9 molecule differs from the sequence of the consensus sequence disclosed in Figure 2 by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 0 amino acid residues. In an embodiment, a Cas9 molecule comprises the amino acid sequence of SEQ ID NO:7 of FIG. 5, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, or N. meningitidis, "-" indicates any amino acid, and "-" indicates any amino acid or absent. In an embodiment, a Cas9 molecule differs from the sequence of SEQ ID NO:6 or 7 by at least , but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0468] A comparison of the sequence of a number of Cas9 molecules indicate that certain regions are conserved. These are identified below as: region 1 (residues 1 to 180, or in the case of region Γ residues 120 to 180) region 2 (residues 360 to 480); region 3 (residues 660 to 720); region 4 (residues 817 to 900); and region 5 (residues 900 to 960). [0469] In an embodiment, a Cas9 molecule comprises regions 1-5, together with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In an embodiment, each of regions 1-6, independently, have, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the corresponding residues of a Cas9 molecule described herein, e.g., a sequence from FIG. 2 or from FIG. 5.

[0470] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1: having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 1-180 (the numbering is according to the motif sequence in FIG. 2; 52% of residues in the four Cas9 sequences in Fig. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes;

differs by at least , 2, 5, 10 or 20 amino acids but by no more than 90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans, L. innocua, N. meningitidis, or S. aureus; or

is identical to 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus,

S. mulans or, L. innocua, N. meningitidis, or S. aureus.

[0471] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region ' : having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 120-180 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans or, L. innocua, N. meningitidis, or S. aureus;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans or, . innocua, N. meningitidis, or S. aureus ; or

is identical to 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans or, L. innocua, N. meningitidis, or S. aureus.

[0472] n an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 2: having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 997c homology with amino acids 360-480 (52% of residues in the four Cas9 sequences in FIG. 2 are

conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, . innocua, N. meningitidis, or S. aureus;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or is identical to 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.

[0473] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 3: having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 660-720 (56% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L: innocua, N. meningitidis, or S. aureus; or is identical to 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.

[0474] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 4: having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 817-900 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus ; or is identical to 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus. [0475] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 5: having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 900-960 (60% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;

differs by at least , 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or is identical to 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus .

A RuvC-like domain and an HNH-like domain

[0476] In an embodiment, a Cas9 molecule comprises an HNH-like domain and an RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH- like domain. A Cas9 molecule, e.g., an eaCas9 or eiCas9 molecule, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In an embodiment, a cas9 molecule is an eaCas9 molecule and the eaCas9 molecule comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below. In an embodiment, a Cas9 molecule is an eiCas9 molecule comprising one or more difference in an RuvC-like domain and/or in an HNH-like domain as compared to a reference Cas9 molecule, and the eiCas9 molecule does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wildype, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1% of the a reference Cas9 molecule, as measured by an assay described herein.

RuvC-like domains [0477] In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non- complementary strand of the target nucleic acid molecule. A Cas9 molecule can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In an embodiment, an RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 8, 17, 16 or 15 amino acids in length. In an embodiment, the cas9 molecule comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.

N-terminal RuvC-like domains [0478] Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain, with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.

[0479] In an embodiment, an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:

D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9 (SEQ ID NO: 8), wherein, X is selected from I, V, M, L and T (e.g., selected from I, V, and L); X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I); X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N); X4 is selected from S, Y, N and F (e.g., S); X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L); X6 is selected from W, F, V, Y, S and' L (e.g., W); X7 is selected from A, S, C, V and G (e.g., selected from A and S); X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ∆ , F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and ∆ ).

[0480] In an embodiment, the N-tenninal RuvC-like domain differs from a sequence of SEQ ID NO:8, by as many as 1 but no more than 2, 3, 4, or 5 residues.

[0481] In embodiment the N-terminal RuvC-like domain is cleavage competent.

[0482] In embodiment the N-terminal RuvC-like domain is cleavage incompetent.

[0483] In an embodiment, an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II: D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9 (SEQ ID NO: 9), wherein

X 1 is selected from I, V, M , L and T (e.g., selected from I, V , and L); X 2 is selected from T , I, V , S , N, Y, E and L (e.g., selected from T , V , and I); X 3 is selected from N , S , G , A , D , T , R , and F (e.g., A or N);

X5 is selected from V , I, L, C , T and F (e.g., selected from V , I and L); X 6 is selected from W , F , V , Y , S and L (e.g., W);

X 7 is selected from A , S , C , V and G (e.g., selected from A and S);

X8 is selected from V , I, L, A , M and H (e.g., selected from V , I, M and L); and

X9 is selected from any amino acid or is absent (e.g., selected from T , V , I, L, ∆ , F , S, A , Y , M and R or selected from e.g., T , V , I, L and ∆).

[0484] In an embodiment, the N-temninal RuvC-like domain differs from a sequence of SEQ ID

NO:9 b y as many as 1, but no more than 2, 3, 4 , or 5 residues.

[0485] n an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula HI:

D-I-G-X2-X3-S-V-G-W-A-X8-X9 (SEQ ID NO: 10), wherein

X 2 is selected from T , I, V , S , N , Y , E and L (e.g., selected from T , V , and I); X3 is selected from N , S, G , A , D , T , R, M and F (e.g., A or N);

X 8 is selected from V , I, L, A , M and H (e.g., selected from V , I, M and L); and

X9 is selected from any amino acid or is absent (e.g., selected from T , V , I, L, ∆ , F, S , A , Y , M and R or selected from e.g., T , V , I, L and ∆).

[0486] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID

NO: 10 by as many as 1, but no more than, 2, 3, 4 , or 5 residues.

[0487] In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence o f formula 11:

D-I-G-T-N-S-V-G-W-A-V-X (SEQ ID NO: 1 ), wherein

X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V , I, L and T (e.g., the eaCas9 molecule can comprise an N-terminal RuvC-like domain shown in FIG. 2 (depicted as "Y")).

[0488] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 1 by as many as 1but no more than, 2, 3, 4, or 5 residues.

[0489] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-

terminal RuvC-like domain disclosed herein, e.g., in FIG. 3A or FIG. 5, as many as 1, but no more than 2, 3, 4, or 5 residues. In an embodiment, , 2, or all 3 of the highly conserved residues identified in FIG. 3A or FIG. 5 are present.

[0490] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-

terminal RuvC-like domain disclosed herein, e.g., in FIG. 3B, as many as 1, but no more than 2,

3, 4, or 5 residues. In an embodiment, 1, 2, 3 or all 4 of the highly conserved residues identified in FIG. 3B are present.

Additional RuvC-like domains

[0491] In addition to the N-terminal RuvC-like domain, a Cas9 molecule, e.g., an eaCas9 molecule, can comprise one or more additional RuvC-like domains. In an embodiment, a Cas9 molecule can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like

domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to

10 amino acids in length, e.g., 8 amino acids in length. [0492] An additional RuvC-like domain can comprise an amino acid sequence:

I-X1-X2-E-X3-A-R-E (SEQ ID NO: 12), wherein X is V or H,

X2 is I, L or V (e.g., I or V); and X3 is or T.

[0493] In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:

I-V-X2-E-M-A-R-E (SEQ ID NO: 13), wherein

X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule can comprise an additional RuvC- like domain shown in FIG. 2 or FIG. 5 (depicted as "B")).

[0494] An additional RuvC-like domain can comprise an amino acid sequence: H-H-A-X 1-D-A-X2-X3 (SEQ ID NO: 14), wherein

I is H o r L;

X 2 is R o r V ; and

X 3 is E o r V .

[0495] In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:.

H-H-A-H-D-A-Y-L (SEQ ID NO: 15).

[0496] In an embodiment, the additional RuvC-like domain differs from a sequence o f SEQ ID

NO: 13 , 15 , 12 o r 14 by as many as 1, but no more than 2 , 3 , 4 , o r 5 residues.

[0497] In some embodiments, the sequence flanking the N-terminal RuvC-like domain is a sequences o f formula V :

-X -Y-X2 -X3'-X4' -Z-T-D-X9'-Y (SEQ ID NO: 16),

wherein

X V is selected from and P,

X2' is selected from V , L, I, and F (e.g., V , I and L);

X3' is selected from G , A and S (e.g., G),

X4' is selected from L, I, V and F (e.g., L);

X9' is selected from D , E, N and Q ; and

Z is an N-terminal RuvC-like domain, e.g. , as described above.

HN H-like domains

[0498] In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand o f a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15 , 20, 25 amino acids in length but not more than

40, 3 5 o r 3 0 amino acids in length, e.g., 20 to 3 5 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.

[0499] In an embodiment, an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VI: 1-X2-X3-H-X4-X5-P-X6-X7-X8-X9-X 10-X 1 -X 12-X 13-X 4-X 15-N-X 6-X 17-X 18- X 19-X20-X21-X22-X23-N (SEQ ID NO: 17), wherein

X is selected from D, E, Q and N (e.g., D and E); X2 is selected from L, I, R, Q, V, M and ; X3 is selected from D and E;

X4 is selected from I, V, T, A and L (e.g., A, 1and V); X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

X6 is selected from Q, H, R, , Y, I, L, F and W; X7 is selected from S, A, D, T and K (e.g., S and A); X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F); X9 is selected from L, R, T, I, V, S, C, Y, , F and G; X 0 is selected from , Q, Y, T, F, L, W, M, A, E, G, and S; X I is selected from D, S, N, R, L and T (e.g., D); X12 is selected from D, N and S;

X 13 is selected from S, A, T, G and R (e.g., S);

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);

X 1 is selected from D, S, 1, N, E, A, H, F, L, Q, M, G, Y and V;

X 16 is selected from K, L, R, M, T and F (e.g., L, R and K); X17 is selected from V, L, I, A and T;

X 8 is selected from L, I, V and A (e.g., L and I); X19 is selected from T, V, C, E, S and A (e.g., T and V); X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A; X21 is selected from S, P, R, , N, A, H, Q, G and L;

X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

[0500] In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 17 by at

least 1, but no more than, 2, 3, 4, or 5 residues.

[0501] In an embodiment, the HNH-like domain is cleavage competent.

[0502] In an embodiment, the HNH-like domain is cleavage incompetent. [0503] In an embodiment, an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII: X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-V-L-X19-X20-X21- X22-X23-N (SEQ ID NO: 18), wherein XI is selected from D and E; X2 is selected from L, I, R, Q, V, M and K; X3 is selected from D and E; X4 is selected from I, V, T, A and L (e.g., A, I and V);

X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

X6 is selected from Q, H, R, K, Y, I, L, F and W;

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F); . X9 is selected from L, R, T, I, V, S, C, Y, K, F and G; X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S; X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F); X is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V; X19 is selected from T, V, C, E, S and A (e.g., T and V); X20 is selected from R, F, T, W, E, L, N, C, , V, S, Q, I, Y, H and A; X21 is selected from S, P, R, K, N, A, H, Q, G and L; X22 is selected from D, G, T, N, S, , A, I, E, L, Q, R and Y; and X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

[0504] In an embodiment, the HNH-like domain differs from a sequence of SEQ ID

NO: 8 by 1, 2, 3, 4, or 5 residues.

[0505] In an embodiment, an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII: X 1-V-X3-H-I- V-P-X6-S-X8-X9-X 10-D-D-S-X 14-X 15-N-K-V-L-T-X20-X2 -X22-X23- N (SEQ ID NO: 19), wherein X 1 is selected from D and E; X3 is selected from D and E; X6 is selected from Q, H, R, K, Y, I, L and W; X8 is selected from F, L, V, , Y, M, I, R, A, E, D and Q (e.g., F); X9 is selected from L, R, T, I, V, S, C, Y, , F and G; X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S; 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F); X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A; X21 is selected from S, P, R, K, N, A, H, Q, G and L; X22 is selected from D, G, T, N, S, , A, I, E, L, Q, R and Y; and

X23 is selected from , V, A, E, Y, I, C, L, S, T, G, , M, D and F.

[0506] In an embodiment, the HNH-like domain differs from a sequence of SEQ D NO: 19 by 1, 2, 3, 4, or 5 residues.

[0507] n an embodiment, an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VIII:

D-X2-D-H-I-X5-P-Q-X7-F-X9-X10-D-X12-S-I-D-N-X16-V-L-X19-X20-S-X22-X23-N (SEQ ID NO:20), wherein X2 is selected from I and V; X5 is selected from I and V; X7 is selected from A and S; X9 is selected from I and L; X 10 is selected from K and T; X12 is selected from D and N; X16 is selected from R, and L; X19 is selected from T and V; X20 i selected from S and R; X22 is selected from K, D and A; and X23 is selected from E, K, G and N (e.g., the eaCas9 molecule can comprise an HNH- like domain as described herein).

[0508] In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO:20 by as many as 1, but no more than 2, 3, 4, or 5 residues. [0509] In an embodiment, an eaCas9 molecule comprises the amino acid sequence of formula IX: L-Y-Y-L-Q-N-G-X1'-D-M-Y-X2'-X3'-X4'-X5'-L-D-I— X6'-X7'-L-S-X8'-Y-Z-N-R- X9'-K-X10'-D-X1 '-V-P (SEQ ID NO:21), wherein X ' is selected from K and R; X2' is selected from V and T; X3' is selected from G and D; X4' is selected from E, Q and D; X5' is selected from E and D; X6' is selected from D, N and H; X7' is selected from Y, R and N; X8' is selected from Q, D and N; X9' is selected from G and E; X10' is selected from S and G; X I 1' is selected from D and N; and Z is an HNH-like domain, e.g., as described above.

[0510] In an embodiment, the eaCas9 molecule comprises an amino acid sequence that differs from a sequence of SEQ ID NO:21 by as many as 1, but no more than 2, 3, 4, or 5 residues.

[0511] In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4A or FIG. 5, as many as 1, but no more than 2, 3, 4, or 5 residues.

[0512] In an embodiment, the HNH -like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4B, by as many as 1, but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, all 3 of the highly conserved residues identified in FIG. 4B are present.

Altered Cas9 Molecules

[0513] Naturally occurring Cas9 molecules possess a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). n an embodiment, a Cas9 molecules can include all or a subset of these properties. In typical embodiments, Cas9 molecules have the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules.

[0514] Cas9 molecules with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecules to provide an altered Cas9 molecule having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule can be introduced. Such mutations and differences comprise: substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In an embodiment, a Cas9 molecule can comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference Cas9 molecule.

[0515] In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, exemplary activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in: one or more RuvC-like domain, e.g., an N- terminal RuvC-like domain; an HNH-like domain; a region outside the RuvC-like domains and the HNH-like domain. In some embodiments, a mutation(s) is present in an N-terminal RuvC- like domain. In some embodiments, a mutation(s) is present in an HNH-like domain. In some embodiments, mutations are present in both an N-terminal RuvC-like domain and an HNH-like domain.

[0516] Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc, can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative or by the method described in Section ΠΙ. In an embodiment, a "non-essential" amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an "essential" amino acid residue results in a substantial loss of activity (e.g., cleavage activity).

[0517] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2 or SEQ ID NO:7. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.

[0518] In an embodiment, the altered Cas9 molecule comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in

Figure 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;

the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 0, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule.

[0519] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.

[0520] In an embodiment the altered Cas9 molecule comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in

Figure 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;

the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-

" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule.

[0521] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. mutans (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.

[0522] In an embodiment the altered Cas9 molecule comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;

the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than , 2, 3, 4, 5, 0, , 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutatis Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-

" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutatis Cas9 molecule.

[0523] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of L. intiocula shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of L. innocula (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of L. innocula shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.

[0524] In an embodiment the altered Cas9 molecule comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in

Figure 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-

" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule.

[0525] In an embodiment, the altered Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule, can be a fusion, e.g., of two of more different Cas9 molecules, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of Cas9 of S. pyogenes comprising an N-terminal

RuvC-like domain can be fused to a fragment of Cas9 of a species other than S. pyogenes (e.g., S. thermophiliis) comprising an HNH-like domain.

Cas9 Molecules with altered PAM recognition or no PAM recognition [0526] Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for S. pyogenes, S. thermophilus,S. mutans, S. aureus and N. meningitidis.

[0527] In an embodiment, a Cas9 molecule has the same PAM specificities as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity to decrease off target sites and increase specificity. In

an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules that recognize different PAM sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt el ai, Nature 201 1, 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section III.

Non-Cleaving and Modified-Cleavage Cas9 Molecules

[0528] In an embodiment, a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

Modified Cleavage eaCas9 Molecules

[0529] In an embodiment, an eaCas9 molecule comprises one or more of the following activities: cleavage activity associated with an N-terminal RuvC-like domain; cleavage activity associated with an HNH-like domain; cleavage activity associated with an HNH domain and cleavage activity associated with an N-terminal RuvC-like domain.

[0530] In an embodiment an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:2 1) and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N- terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figure 2 or an

aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the N-terminal RuvC-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 0, 5, 1 or 0. 1 % of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. ihermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.

[0531] In an embodiment, an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, N-terminal RuvC-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 1 , SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine at position 856 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in Figure 2 and/or at position 879 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the HNH- like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as

a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.

Non-Cleaving eiCas9 Molecules

[0532] In an embodiment, the altered Cas9 molecule is an eiCas9 molecule which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% o the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9

molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of . . pyogenes, S.

thermophilus, S. aureus or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule lacks substantial cleavage activity associated with an N- terminal RuvC-like domain and cleavage activity associated with an HNH-like domain.

[0533] In an embodiment, an eiCas9 molecule comprises an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figure 2 or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine.

[0534] In an embodiment an eiCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 5). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine at position 856 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in Figure 2 and/or at position 879 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine.

[0535] A catalytically inactive Cas9 molecule may be fused with a transcription repressor. An eiCas9 fusion protein complexes with a gRNA and localizes to a DNA sequence specified by gRNA's targeting domain, but, unlike an eaCas9, it will not cleave the target DNA. Fusion of an effector domain, such as a transcriptional repression domain, to an eiCas9 enables recmitment of the effector to any DNA site specified by the gRNA. Site specific targeting of an eiCas9 or an eiCas9 fusion protein to a promoter region of a gene can block R A polymerase binding to the promoter region, a transcription factor (e.g., a transcription activator) and/or a transcriptional enhancer to inhibit transcription activation. Alternatively, site specific targeting of an eiCas9- fusion to a transcription repressor to a promoter region of a gene can be used to decrease transcription activation.

[0536] Transcription repressors or transcription repressor domains that may be fused to an eiCas9 molecule can include Kruppel associated box (KRAB or SKD), the Mad mSIN3 interaction domain (SID) or the ERF repressor domain (ERD).

[0537] In another embodiment, an eiCas9 molecule may be fused with a protein that modifies chromatin. For example, an eiCas9 molecule may be fused to heterochromatin protein 1 (HPl), a histone lysine methyltransferase (e.g., SUV39H1, SUV39H2, G9A, ESET/SETDBl, Pr-SET7/8, SUV4-20H1, RIZ1), a histone lysine demethylates (e.g., LSD1/BHC1 10, SpLsdl/Sw,l/Safl 10, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, Rphl, JARID1A/RBP2, JARIDIB/PLU-I, JAR1D1C/SMCX, JARID1D/SMCY, Lid, Jhn2, Jmj2), a histone lysine deacetylases (e.g., HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hosl, Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hdal, Cir3, SIRT1, SIRT2, Sir2, Hstl, Hst2, Hst3, Hst4, HDAC1 ) and a DNA methylases (DNMT1 , DNMT2a/DMNT3b, MET1). An eiCas9-chomatin modifying molecule fusion protein can be used to alter chromatin status to reduce expression a target gene.

[0538] The heterologous sequence (e.g., the transcription repressor domain) may be fused to the N- or C-terminus of the eiCas9 protein. n an alternative embodiment, the heterologous sequence (e.g., the transcription repressor domain) may be fused to an internal portion (i.e., a portion other than the N-terminus or C-terminus) of the eiCas9 protein.

[0539] The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated, e.g., by the methods described herein in Section ΠΙ. The activity of a Cas9 molecule, either an eaCas9 or a eiCas9, alone or in a complex with a gRNA molecule may also be evaluated by methods well-known in the art, including, gene expression assays and chromatin-based assays, e.g., chromatin immunoprecipitation (ChiP) and chromatin in vivo assay (CiA).

Nucleic Acids Encoding Cas9 Molecules

[0540] Nucleic acids encoding the Cas9 molecules, e.g., an eaCas9 molecule or an eiCas9 molecule are provided herein.

[0541] Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al, SCIENCE

2013, 399(61 21):819-823; Wang et al, CELL 2013, 153(4):910-918; Mali et al., SCIENCE 2013,

399(6121):823-826; Jinek et al, SCIENCE 2012, 337(6096):816-821. Another exemplary nucleic acid encoding a Cas9 molecule of N. meningitidis is shown in FIG. 6.

[0542] In an embodiment, a nucleic acid encoding a Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section X. In an embodiment, the Cas9 mRNA has one or more of, e.g., all of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.

[0543] In addition or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. Fo example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.

[0544] In addition, or alternatively, a nucleic acid encoding a Cas9 molecule may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0545] Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S'. pyogenes.

ATGGATAAAA AGTACAGCAT CGGGCTGGAC ATCGGTACAA ACTCAGTGGG GTGGGCCGTG ATTACGGACG AGTACAAGGT ACCCTCCAAA AAATTTAAAG TGCTGGGTAA CACGGACAGA CACTCTATAA AG AAAATCT TATTGGAGCC TTGC TGTTCG ACTCAGGCGA GACAGCCGAA GCCACAAGGT TGAAGCGGAC CGCCAGGAGG CGGTATACCA GGAGAAAGAA CCGCATATGC TACCTGCAAG AAATCTTCAG TAACGAGATG GCAAAGGTTG ACGATAGCTT TTTCCATCGC CTGGAAGAAT. CCTTTCTTGT TGAGGAAGAC AAGAAGCACG AACGGCACCC CATCTTTGGC AATATTGTCG ACGAAGTGGC ATATCACGAA AAGTACCCGA CTATCTACCA CCTCAGGAAG AAGCTGGTGG ACTOTACCGA TAAGGCGGAC CTCAGACTTA TTTATTTGGC ACTCGCCCAC ATGATTAAAT TTAGAGGACA TTTCTTGATC GAGGGCGACC TGAACCCGGA CAACAGTGAC GTCGATAAGC TGT TCATCCA ACTTGTGCAG ACCTACAATC AACTGTTCGA AGAAAACCCT ATAAATGCTT CAGGAGTCGA CGCTAAAGCA ATCCTGTCCG CGCGCCTCTC AAAATCTAGA AGACTTGAGA ATCTGATTGC TCAGTTGCCC GGGGAAAAGA AAAATGGATT GT TTGGCAAC CTGATCGCCC TCAGTCTCGG ACTGACCCCA AATTTCAAAA GTAACTTCGA CCTGGCCGAA GACGCTAAGC TCCAGCTGTC CAAGGACACA TACGATGACG ACCTCGACAA TCTGCTGGCC CAGATTGGGG ATCAGTACGC CGATCTCT TT TTGGCAGCAA AGAACCTGTC CGACGCCATC CTGT TGAGCG ATATCTTGAG AGTGAACACC GAAATTACTA AAGCACCCCT TAGCGCATCT ATGATCAAGC GGTACGACGA GCATCATCAG GATCTGACCC TGC TGAAGGC TCTTGTGAGG CAACAGCTCC CCGAAAAATA CAAGGAAATC TTCT TTGACC AGAGCAAAAA CGGCTACGCT GGCTATATAG ATGGTGGGGC GAGTCAGGAG GAATTCTATA AATTCATCAA GCCCATTCTC GAGAAAATGG ACGGCACAGA GGAGTTGCTG GTCAAACTTA ACAGGGAGGA CCTGCTGCGG AAGCAGCGGA CCTTTGACAA CGGGTCTATC CCCCACCAGA TTCATCTGGG CGAACTGCAC GCAATCCTGA GGAGGCAGGA GGATTTTTAT CCTTTTCTTA AAGATAACCG CGAGAAAATA GAAAAGATTC TTACATTCAG GATCCCGTAC TACGTGGGAC CTCTCGCCCG GGGCAATTCA CGGTTTGCCT GGATGACAAG GAAGTCAGAG GAGACTATTA CACCTTGGAA CTTCGAAGAA GTGGTGGACA AGGGTGCATC TGCCCAGTC T TTCATCGAGC GGATGACAAA TTTTGACAAG AACCTCCCTA ATGAGAAGGT GCTGCCCAAA CATTCTCTGC TCTACGAGTA CTTTACCGTC TACAATGAAC TGACTAAAGT CAAGTACGTC ACCGAGGGAA TGAGGAAGCC GGCATTCCTT AGTGGAGAAC AGAAGAAGGC GATTGTAGAC CTGTTGTTCA AGACCAACAG GAAGGTGACT GTGAAGCAAC TTAAAGAAGA CTAC TTTAAG AAGATCGAAT GTTT TGACAG TGTGGAAATT TCAGGGGTTG AAGACCGCTT CAATGCGTCA TTGGGGACTT ACCATGATC T TCTCAAGATC ATAAAGGACA AAGACTTCCT GGACAACGAA GAAAATGAGG ATATTCTCGA AGACATCGTC CTCACCCTGA CCCTGTTCGA AGACAGGGAA ATGATAGAAG AGCGCTTGAA AACCTATGCC CACCTCTTCG ACGATAAAGT TATGAAGCAG CTGAAGCGCA GGAGATACAC AGGATGGGGA AGATTGTCAA GGAAGCTGAT CAATGGAATT AGGGATAAAC AGAGTGGCAA GACCATACTG . GATTTCCTCA AATCTGATGG CTTCGCCAAT AGGAACTTCA TGCAACTGAT TCACGATGAC TCTCTTACCT TCAAGGAGGA CATTCAAAAG GCTCAGGTGA GCGGGCAGGG AGACTCCCTT CATGAACACA TCGCGAATTT GGCAGGTTCC CCCGCTATTA AAAAGGGCAT CCTTCAAACT GTCAAGGTGG TGGATGAATT GGTCAAGGTA ATGGGCAGAC ATAAGCCAGA AAATATTGTG ATCGAGATGG CCCGCGAAAA CCAGACCACA CAGAAGGGCC AGAAAAATAG TAGAGAGCGG ATGAAGAGGA TCGAGGAGGG CATCAAAGAG CTGGGATCTC AGATTCTCAA AGAACACCCC GTAGAAAACA CACAGCTGCA GAACGAAAAA TTGTACTTGT ACTATCTGCA GAACGGCAGA GACATGTACG TCGACCAAGA ACTTGATATT AATAGACTGT CCGAC TATGA CGTAGACCAT ATCGTGCCCC AGTCCTTCCT GAAGGACGAC TCCAT TGATA ACAAAGTCTT GACAAGAAGC GACAAGAACA GGGGTAAAAG TGATAATGTG CCTAGCGAGG AGGTGGTGAA AAAAATGAAG AACTACTGGC GACAGCTGCT TAATGCAAAG CTCATTACAC AACGGAAGTT CGATAATCTG ACGAAAGCAG AGAGAGGTGG CTTGTCTGAG TTGGACAAGG- CAGGGTTTAT TAAGCGGCAG CTGGTGGAAA CTAGGCAGAT CACAAAGCAC GTGGCGCAGA TTTTGGACAG CCGGATGAAC ACAAAATACG ACGAAAATGA TAAACTGATA CGAGAGGTCA AAGTTATCAC GCTGAAAAGC AAGCTGGTGT CCGATTTTCG GAAAGACTTC CAGTTCTACA AAGTTCGCGA GATTAATAAC TACCATCATG CTCACGATGC GTACCTGAAC GCTGTTGTCG GGACCGCCTT GATAAAGAAG TACCCAAAGC TGGAATCCGA GTTCGTATAC GGGGATTACA AAGTGTACGA TGTGAGGAAA ATGATAGCCA AG.TCCGAGCA GGAGATTGGA AAGGCCACAG CTAAGTACTT CTTTTATTCT AACATCATGA ATTTTTTTAA GACGGAAATT ACCCTGGCCA ACGGAGAGAT CAGAAAGCGG CCCCTTATAG AGACAAATGG TGAAACAGGT GAAATCGTCT GGGATAAGGG CAGGGATTTC GCTACTGTGA GGAAGGTGCT GAGTATGCCA CAGGTAAATA TCGTGAAAAA AACCGAAGTA CAGACCGGAG GATTTTCCAA GGAAAGCATT TTGCCTAAAA GAAACTCAGA CAAGCTCATC GCCCGCAAGA AAGATTGGGA CCCTAAGAAA TACGGGGGAT TTGACTCACC CACCGTAGCC TATTCTGTGC TGGTGGTAGC TAAGGTGGAA AAAGGAAAGT CTAAGAAGCT GAAGTCCGTG AAGGAACTCT TGGGAATCAC TATCATGGAA AGATCATCCT T GAAA G CCCTATCGAT TTCCTGGAGG CTAAGGGTTA CAAGGAGGTC AAGAAAGACC TCATCATTAA ACTGCCAAAA TACTCTCTCT TCGAGCTGGA AAATGGCAGG AAGAGAATGT TGGCCAGCGC CGGAGAGCTG CAAAAGGGAA ACGAGCTTGC TCTGCCCTCC AAATATGTTA ATTTTC TCTA TCTCGCTTCC CACTATGAAA AGCTGAAAGG GTC TCCCGAA GATAACGAGC AGAAGCAGCT GTTCGTCGAA CAGCACAAGC ACTATCTGGA TGAAATAATC GAACAAATAA GCGAGTTCAG CAAAAGGGTT ATCCTGGCGG ATGCTAATTT GGACAAAGTA CTGTCTGCTT ATAACAAGCA CCGGGATAAG CCTATTAGGG AACAAGCCGA GAATATAATT CACCTCTTTA CACTCACGAA TCTCGGAGCC CCCGCCGCCT TCAAATACTT TGATACGACT ATCGACCGGA AACGGTATAC CAGTACCAAA GAGGTCCTCG ATGCCACCC T CATCCACCAG TCAATTACTG GCCTGTACGA AACACGGATC GACCTCTCTC AACTGGGCGG CGAC TAG (SEQ D NO: 22)

[0546] Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.

DKKYS I GLD I GTNSVGWAVI TDEYKVPSKKFKVLGNTDRH S I KKNL I GALLFDSGETAEATRL KRTARRRYTRRKNR I CYLQE I F SNEMAKVDDSFFHRLEESFLVEEDKKHERHP I FGN IVDEVAY HEKYPT I YHLRKKLVDS TDKADLRL I YLALAHMI KFRGHFL IEGDLNPDNSDVDKLF QLVQTY NQLFEENP INASGVDAKAI LSARL SKSRRLENL IAQLPGEKKNGLFGNL IAL SLGLTPNFKSNF DLAEDAKLQL SKDTYDDDLDNLLAQ I GDQYADLFLAAKNL SDAI LL SDI LRVNTE I TKAPL SAS MI KRYDEHHQDLTLLKALVRQQLPEKYKE I FFDQSKNGYAGYI DGGASQEEFYKF IKP I LEKMD GTEELLVKLNREDLLRKQRTFDNGS IPHQI HLGELHAI LRRQEDFYPFLKDNREKIEKI LTFRI PYYVGPLARGNSRFAWMTRKSEET I TPWNFEEVVDKGASAQSF IERMTNFDKNLPNEKVLPKH S LLYEYFTVYNEL TKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVE I SGVEDRFNAS LGTYHDLLK I KDKDFLDNEENED I LED IVL TLTLFEDREMI EERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKL I NGI RDKQSGKT I LDFLKS DGFANRNFMQL I HDDS LTF KED I QKAQVSGQGDSLHEH IANLAG SPAI KKG LQTVKVVDELVKVMGRHKPEN I I E ARENQ TTQKGQKNSRERMKRIEEG I KELG SQI LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INR LSDYDVDH IVPQSF LKDDS I DNKVLTRS DKNRGKS DNVPSEEVVKKMKNYWRQLLNAKL TQRK FDNL TKAERGGL SELDKAGF I KRQLVETRQ I TKHVAQI LDSRMNTKYDENDKL I REV VI TLKS KLVS DFRKDFQFYKVRE INNYHHAHDAYLNAVVGTAL I KKYPKLE SEFVYGDYKVYDVRKMI AK SEQE I GKATAKYFFYSN I MNFFKTE I TLANGE I RKRPL I ETNGETGE IVWDKGRDFATVRKVL S MP QV VKKTEVQTGGF SKE S I LPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLG T IMERS SFEKNP DFLEAKGYKEVKKDL I I KLPKYS LFELENGRKRMLAS AGELQKGNELALPS KYVNFLYLAS HYEKLKGSPEDNEQKQLFVEQHKHYLDE I EQI SEF SKRV I LADANLDKVL SAYNKHRDKP IREQAENI I HLFTLTNLGAPAAFKYFDTT I DR RY STKEVLD ATL I HQS TGLYETRI DLSQLGGD * (SEQ ID NO: 23)

[0547] Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitidis.

ATGGCCGCCTTCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACATCGGCATCGCCAGCG TGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGAACCCCATC TGCCTGATCGACCTGGGTGT GCGCGTGTTCGAGCGCGCTGAGGTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCTT GCTCGCTCTGTTCGGCGCCT TAC TCGCCGGCGCGCTCACCGCCTTCTGCGCGCTCGCCGCCTGC TGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACTTCGACGAGAACGGCCTGATCAAGAGCCTGCC CAACACTCCTTGGCAGCTGCGCGCTGCCGCTCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGC GCCGTGCTGCTGCACCTGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGCGAGA CCGCCGACAAGGAGCTGGGTGCTC TGCTGAAGGGCGTGGCCGACAACGCCCACGCCCTGCAGAC TGGTGACTTCCGCACTCCTGCTGAGC TGGCCCTGAACAAGTTCGAGAAGGAGAGCGGCCACATC CGCAACCAGCGCGGCGACTACAGCCACACC TTCAGCCGCAAGGACCTGCAGGCCGAGCTGATCC TGCTGTTCGAGAAGCAGAAGGAGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGGGCAT CGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCCGTGCAGAAGATGCTGGGC CACTGCACCTTCGAGCCAGCCGAGCCCAAGGCCGCCAAGAACACC TACACCGCCGAGCGCT TCA TCTGGCTGACCAAGCTGAACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGACCGA CACCGAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTACGCCCAGGCC CGCAAGCTGCTGGGTCTGGAGGACACCGCCTTCTTCAAGGGCCTGCGCTACGGCAAGGACAACG CCGAGGCCAGCACCCTGATGGAGATGAAGGCCTACCACGCCATCAGCCGCGCCCTGGAGAAGGA GGGCCTGAAGGACAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGGCACC GCCTTCAGCCTGTTCAAGACCGACGAGGACATCACCGGCCGCCTGAAGGACCGCATCCAGCCCG AGATCCTGGAGGCCCTGCTGAAGCACATCAGCTTCGACAAGTTCGTGCAGATCAGCCTGAAGGC CCTGCGCCGCATCGTGCCCCTGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGAGATC TACGGCGACCACTACGGCAAGAAGAACACCGAGGAGAAGATCTACCTGCCTCCTATCCCCGCCG ACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGGCCCGCAAGGTGATCAACGGCGT GGTGCGCCGCTACGGCAGCCCCGCCCGCATCCACATCGAGACCGCCCGCGAGGTGGGCAAGAGC TTCAAGGACCGCAAGGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGGCCG CCGCCAAGTTCCGCGAGTAC TTCCCCAACTTCGTGGGCGAGCCCAAGAGCAAGGACATCCTGAA GC TGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCTGTACAGCGGCAAGGAGATCAACCTGGGC CGCC TGAACGAGAAGGGCTACGTGGAGATCGACCACGCCCTGCCCTTCAGCCGCACCTGGGACG ACAGCTTCAACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCAGACCCC CTACGAGTACTTCAACGGCAAGGACAACAGCCGCGAGTGGCAGGAGTTCAAGGCCCGCGTGGAG ACCAGCCGCTTCCCCCGCAGCAAGAAGCAGCGCATCCTGCTGCAGAAGTTCGACGAGGACGGCT TCAAGGAGCGCAACCTGAACGACACCCGCTACGTGAACCGCTTCCTGTGCCAGTTCGTGGCCGA CCGCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGTTCGCCAGCAACGGCCAGATCACCAAC CTGC TGCGCGGCTTCTGGGGCCTGCGCAAGGTGCGCGCCGAGAACGACCGCCACCACGCCCTGG ACGCCGTGGTGGTGGCCTGCAGCACCGTGGCCATGCAGCAGAAGATCACCCGCTTCGTGCGCTA CAAGGAGATGAACGCCTTCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCACCAG AAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGATGATCCGCGTGTTCGGCA AGCCCGACGGCAAGCCCGAGTTCGAGGAGGCCGACACCCCCGAGAAGCTGCGCACCCTGCTGGC CGAGAAGCTGAGCAGCCGCCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGTTCGTGAGCCGC GCCCCCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCCAAGCGCC TGG ACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCTGAAGCTGAAGGACCTGGAGAAGAT GGTGAACCGCGAGCGCGAGCCCAAGCTGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCACAAG GACGACCCCGCCAAGGCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGCACCC AGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGTGCGCAACCACAA CGGCATCGCCGACAACGCCACCATGGTGCGCGTGGACGTGTTCGAGAAGGGCGACAAGTACTAC CTGGTGCCCATCTACAGCTGGCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTGCAGG GCAAGGACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACT TCAAGTTCAGCCTGCACCC CAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTTCGGCTACTTCGCCAGCTGCCAC CGCGGCACCGGCAACATCAACATCCGCATCCACGACCTGGACCACAAGATCGGCAAGAACGGCA TCCTGGAGGGCATCGGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGG CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA (SEQ ID NO: 24)

[0548] Provided below is the corresponding amino acid sequence of a N. meningitidis Cas9 molecule.

MAAFKPNP INY LGLD I G SVG A VE I DEDENP I CL DLGVRVFERAEVPKTGDS LAMARRL ARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGL I KS LPNTPWQLRAAALDRKLTPLEWS AVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHI RNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLG HCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGT AFSLFKTDEDITGRLKDRIQPEILEALLKHI SFDKFVQI SLKALRRIVPLMEQGKRYDEACAEI YGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKS FKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVE TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITN LLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQ KTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSR APNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHK DDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCH RGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR* (SEQ ID NO: 25)

[0549] Provided below is an amino acid sequence of a S. aureus Cas9 molecule.

MKRNYILGLDIGITSVGYGI IDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDT GNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGS INRFKTSDYVKEAKQLLKVQKAYHQ LDQSFI DTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALNDLNNLVITRDENEKLEYYEKFQI IENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK PEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTI YQSSEDIQEELTNLNSELTQEEIEQI S NLKGYTGTHNLSLKAINLILDELWHTNDNQI AIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP VVKRSFIQSIKVINAI IKKYGLPNDI IIELAREKNSKDAQKMINEMQKRNRQTNERIEEI IRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVK QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAED ALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKD YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA EFI ASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKT QSIKKYSTDILGNLYEVKSKKHPQI IKKG* (SEQ ID NO: 26)

[0550] If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C- temninus (e.g., an eiCas9 fused with a transcripon repressor at the C-terminus), it is understood that the stop codon will be removed.

Other Cas Molecules [0551] Various types of Cas molecules can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of Type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et ai,

PLoS COMPUTATIONALBIOLOGY 2005, 1(6): e60 and Makarova et al, NATURE REVIEW

MICROBIOLOGY 201 1, 9:467-477, the contents of both references are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table -1. Tabic ΙΙ· 1: Ca Systems

( i i c System type Nain from Haft Structure of Families (and Representatives name* or subtype et al. encoded protein siiperl'amily) of (P encoded accessions) 1 protein

cas6 • Subtype 1- cas6 and cinx6 314H COG 1583 and PF1 13 1 and slr7014 A COG555 1 • Subtype I- (RAMP)

• Subtype I- D • Subtype ΙΙΙ -Α · Subtype III- B cas6e • Subtype I- cse3 1 J9 (RAMP) ygcH E cas6f • Subtype I- csy4 2XL.I (RAMP) y l 27 F cas7 • Subtype I- csa2, csJ2, cst'4, NA COG 1857 and d v and ygc.l A csh2, cspl and COG3649 • Subtype I- csl2 (RAMP) B . · Subtype I- C • Subtype I- E c.asHa • Subtype I- cinxl, c.stl , x , NA BH0338-like LA3 19 1 and A** csx 13 and PG201 8 cxxc-cxxc cas8a2 • Subtype I- csa4 and csx9 NA PH0918 AF0070, AF1873, A** MJ0385, PF0637, PH09 18 nd SSO 140 1 casSb • Subtype I- cshl and NA BH0338-like MTH 1090 and B** TM 1802 TM 1802

sS • Subtype 1- csdl and csp2 NA BH0338-like B 10338 C** cas9 • Type II** csnl and csx J2 NA COG35 13 FTN_0757 and SPyl046 cas 10 • Type III** cmr2, csml and NA COG 1353 Μ Ί Ή 326, Rv2823c csx 1 and TM1 794 r.aslOd • Subtype I- c.sc.3 NA COG 1353 slr701 D** csyJ • Subtype - csyJ NA y l724-like y l724 F** csy2 • Subtype I- csy2 NA (RAMP) l725 F csy3 • Subtype I- csy3 NA (RAMP) y 726 Table I-l : Cas Systems

G n .System type Name from Waft Structure of Families (and RepresentativGs na * or subtype el al encoded protein supcrlamily) of (P encoded accessions) 1 protein

csel • Subtype I- csel NA YgcL-like ygcL E cse2 • Subtype I- cse2 2ZCA YgcK-like ygcK E cscl • Subtype I- cscl NA alrl 563-like air 1563 n (RAMP) c.sc.2 • Subtype I- cscl and csc2 NA COG 1337 slr70 12 D (RAMP) csa5 • Subtype I- csa5 NA AF1 870 A I870, MJ0380. Λ PF0643 and SSO1 3 8 csn2 • Sublype II- c n2 NA SPyl 049-like SPyl049 A csm2 • Subtype csm2 NA COG 1421 MTH 08 and II1-A" SERP2460 csin3 • Subtype csc2 a d csm3 NA COG 1337 MTH 1080 and Ι-Α (RAMP) SERP2459 csin4 • Subtype csm4 NA COG 1567 MTH 1079 and III-A (RAMP) SERP2458 • Sublype csniS NA COG 332 MTH 1078 and III-A (RAMP) SERP2457 csni6 • Sublype ΛΡΕ 22 6 and 2WTE COG 1 17 ΛΡΕ 2256 and III-A cs 6 SS0 1445 irl • Sublype c.mrl NA COG 1367 P 130 III-B (RAMP) cinr3 • Sublype cmr3 NA COG 1769 PF1 128 III-B (RAMP) cmr4 • Subtype cmr4 NA COG 1336 PF1 126 III-B (RAMP) c nr • Sublype c.mr5 2ZOP and 20EB COG3337 MTH324 and PF1 25 III-B** cmr6 • Subtype cinr6 NA COG 1604 PF1 124 III-B (RAMP) / csbl • Subtype - GSU0053 NA (RAMP) Balac_ 1306 and U GSU0053 csb2 • Sublype 1- NA NA (RAMP) Balac_1 305 and GSU0054 csb3 • Subtype I- NA NA (RAMP) Balac_1 303 U c x 17 • Subtype I- NA NA NA t _2683 Table II- 1: Cas Systems

Genc System type Name fr m II ift Structure of Families (and Representatives Vnaiiie* or subtype et al. encoded protein supcrfamily) of (PDB encoded accessions) 1 protein

csx14 • Subtype 1- NA NA NA GSU0052 U cs.\10 • Subtype I- csx10 NA (RAMP) Caur_2274 U csx16 • Subtype VVA 1548 NA NA VVA1548 -U csaX • Subtype. csaX NA NA SS0 1438 III-U csx3 • Subtype csx3 NA NA AF1 864 III-U csxl • Subtype csa3, csxl. csx2, XMX and 217 1 COG 15 17 and M.I 1666, NE0 113. III-U DXTHG. COG4006 PF1 127 n T 18 12 NE0 113 and TIGR027 10 csx 15 • Unknown NA NA TTE2665 ΤΓΕ 2665 csfl • Type U csfl NA NA AFE_1038 csfl • Type U sfl NA (RAMP) AFE_1039 cs/3 • Type U csf3 NA (RAMP) AFE_1040 cs/4 • Type U csf4 NA NA AFE_1037

III. Functional Analysis of Candidate Molecules

[0552] Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are

described, e.g., in Jinek el ciL , SCIENCE 20 12 ; 337(6096):8 16-82 1.

Binding and Cleavage Assay: Testing the endonuclease activity of Cas9 molecule

[0553] The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target

nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro- transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95°C and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (~8 nM)) is incubated for 60 min at 37°C with purified Cas9 protein molecule (50-500 nM) and

gRNA (50-500 nM, 1: 1) in a Cas9 plasmid cleavage buffer (20 M HEPES pH 7.5, 150 mM

KC1, 0.5 mM DTT, 0 . mM EDTA) with or without 10 mM MgCl 2. The reactions are stopped with 5X DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.

[0554] Alternatively, the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this

assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with .5 units T4 polynucleotide kinase and -3-6 pmol (-20-40 mCi) [γ -32Ρ]-ΑΤΡ in IX T4 polynucleotide

kinase reaction buffer at 37°C for 30 min, in a 50 µΙ_ reaction. After heat inactivation (65°C for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates ( 100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95°C for 3 min, followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95°C for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) is pre- incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM

HEPES pH 7.5, 100 mM C 1, 5 mM MgC12, 1 mM DTT, 5% glycerol) in a total volume of 9 µΐ .

Reactions are initiated by the addition of 1 µΐ target DNA (10 nM) and incubated for 1 h at 37°C.

Reactions are quenched by the addition of 20 µΐ of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95°C for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging. The resulting cleavage products indicate that whether the complementary strand, the non- complementary strand, or both, are cleaved.

[0555] One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule or candidate Cas9 molecule. Binding Assay: Testing the binding of Cas9 molecule to target DNA

[0556] Exemplary methods for evaluating the binding of Cas9 molecule to target DNA are described, e.g., in Jinek et al, SCIENCE 2012; 337(6096):8 16-821.

[0557] For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand (10 nmol) in deionized water, heating to 95°C for 3 min and slow cooling to room temperature. All DNAs are purified on 8% native gels containing IX TBE. DNA bands are visualized by V shadowing, excised, and eluted by soaking gel pieces in

DEPC-treated H20 . Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H 0 . DNA samples are 5' end labeled with [γ -32Ρ]-ΑΤΡ using T4 polynucleotide kinase for 30 min at 37°C. Polynucleotide kinase is heat denatured at 65°C for 20 min, and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KC1, 5 mM MgCl , 1 mM DTT and 10% glycerol in a total volume of 0 µΐ . Cas9 protein molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 pM to 1 µΜ . Radiolabeled DNA is added to a final concentration of 20 pM. Samples are incubated for 1 h at 37°C and resolved at 4°C on an 8% native polyacrylamide gel containing 1X TBE and 5 mM MgCl . Gels are dried and DNA visualized by phosphorimaging.

IV. Template Nucleic Acids (Genome Editing Approaches)

The terms "template nucleic acid" and "swap nucleic acid" are used interchangeably and have identical meaning in this document and its priority documents.

[0558] Mutations in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-

13, VH- 14, VII- 15, VII- 16, V - 17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII- 25, IX- , IX- 1A, D<-2, IX-3, XIV- 1, or Section VIII, may be corrected using one of the approaches discussed herein. In an embodiment, a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VH-14, VII-15, VII- 16, VII-17, VII-18, VII-

19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-1A, IX-2, IX-3, XIV- 1, or Section VIII, is corrected by homology directed repair (HDR) using a template nucleic acid (see Section

IV. 1). In an embodiment, a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII-14, VII-15, VII- 16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-

22, VII-23, VII-24, VII-25, IX- 1, IX- 1A, IX-2, IX-3, XIV- 1, or Section VIII, is corrected by Non-Homologous End Joining (NHEJ) repair using a template nucleic acid (see Section IV. ).

IV.l HDR Repair and Template Nucleic Acids

[0559] As described herein, nuclease-induced homology directed repair (HDR) can be used to alter a target sequence and correct (e.g., repair or edit) a mutation in the genome. W ile not wishing to be bound by theory, it is believed that alteration of the target sequence occurs by homology-directed repair (HDR) with a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target sequence. It is contemplated that a plasmid donor can be used as a template for homologous recombination. It is further contemplated that a single stranded donor template can be used as a template for alteration of the target sequence by alternate methods of homology directed repair (e.g., single strand annealing) between the target sequence and the donor template. Donor template-effected alteration of a target sequence depends on cleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a double strand break or two single strand breaks.

[0560] In an embodiment, a mutation can be corrected by either a single double-strand break or two single strand breaks. In an embodiment, a mutation can be corrected by (1) a single double- strand break, (2) two single strand breaks, (3) two double stranded breaks with a break occurring on each side of the target sequence, (4) one double stranded breaks and two single strand breaks with the double strand break and two single strand breaks occurring on each side of the target sequence or (5) four single stranded breaks with a pair of single stranded breaks occurring on each side of the target sequence.

Double strand break mediated correction

[0561] In an embodiment, double strand cleavage is effected by a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-termina] RuvC-like domain, e.g., a wild type Cas9. Such embodiments require only a single gRNA. Single strand break mediated correction

[0562] In other embodiments, two single strand breaks, or nicks, are effected by a Cas9 molecule having nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments require two gRNAs, one for placement of each single strand break. In an embodiment, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand to which the gRNA hybridizes. In an embodiment, the Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes.

[0563] In an embodiment, the nickase has HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D IOA mutation.

DIOA inactivates RuvC; therefore, the Cas9 nickase has (only) HNH activity and will cut on the strand to which the gRNA hybridizes (e.g., the complementary strand, which does not have the NGG PAM on it). In other embodiments, a Cas9 molecule having an H840, e.g., an H840A, mutation can be used as a nickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (e.g., the strand that has the NGG PAM and whose sequence is identical to the gRNA).

[0564] In an embodiment, in which a nickase and two gRNAs are used to position two single strand nicks, one nick is on the + strand and one nick is on the - strand of the target nucleic acid. The PAMs are outwardly facing. The gRNAs can be selected such that the gRNAs are separated by, from about 0-50, 0- 100, or 0-200 nucleotides. In an embodiment, there is no overlap between the target sequence that is complementary to the targeting domains of the two gRNAs. In an embodiment, the gRNAs do not overlap and are separated by as much as 50, 100, or 200 nucleotides. In an embodiment, the use of two gRNAs can increase specificity, e.g., by decreasing off-target binding (Ran el cil., CELL 20 13).

[0565] In an embodiment, a single nick can be used to induce HDR. It is contemplated herein that a single nick can be used to increase the ratio of HR to NHEJ at a given cleavage site. Placement of the double strand break or a single strand break relative to target position

[0566] The double strand break or single strand break in one of the strands should be sufficiently close to target position such that correction occurs. In an embodiment, the distance is not more than 50, 100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound by theory, it is believed that the break should be sufficiently close to target position such that the break is within the region that is subject to exonuclease-mediated removal during end resection. f the distance between the target position and a break is too great, the mutation may not be included in the end resection and, therefore, may not be corrected, as donor sequence may only be used to correct sequence within the end resection region.

[0567] In an embodiment, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease induce a double strand break for the purpose of inducing HDR-mediated correction, the cleavage site is between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position. In an embodiment, the cleavage site is between 0- 100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.

[0568] In an embodiment, in which two gRNAs (independently, unimolecular (or chimeric) or modular gRNA) complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing HDR-mediated correction, the closer nick is between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position and the two nicks will ideally be within 25-55 bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 bp away from each other). In an embodiment, the cleavage site is between 0- 100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position. [0569] In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the target positionand the second gRNA is used to target downstream (i.e., 3') of the target position). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the target position and the second gRNA is used to target downstream (i.e., 3') of the target position). The double strand break(s) or the closer of the two single strand nicks in a pair will . ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200,

150, lOO, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30,

50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35. to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

[0570] In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17, VII- 18, VII-19, VII-

20, VII-21 , VII-22, VII-23, VU-24, VII-25, IX-1, IX-1 A, IX-2, IX-3, XIV- , or Section VIII and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII-15, VII-16, VII-17,

VII- 18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX-1, IX-1 A, IX-2, IX-3, XIV- 1, o Section VIII). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

Length of the homology arms

[0571] The homology arm should extend at least as far as the region in which end resection may occur, e.g., in order to allow the resected single stranded overhang to find a complementary region within the donor template. The overall length could be limited by parameters such as plasmid size or viral packaging limits. In an embodiment, a homology arm does not extend into repeated elements, e.g., ALU repeats, LINE repeats.

[0572] Exemplary homology arm lengths include a least 50, 100, 250, 500, 750 or 1000 nucleotides.

[0573] Target position, as used herein, refers to a site on a target nucleic acid (e.g., the chromosome) that is modified by a Cas9 molecule-dependent process. For example, the target position can be a modified Cas9 molecule cleavage of the target nucleic acid and template nucleic acid directed modification, e.g., correction, of the target position. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides is added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a target sequence (e.g., the sequence to which the gRN A binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).

[0574] A template nucleic acid, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 molecule and a gRNA molecule to alter the structure of a target position. In an embodiment, the target nucleic acid is modified to have some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the tempolate nuceic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA.

[0575] In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid.

[0576] Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.

[0577] In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation.

[0578] In other embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5' or 3' non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.

[0579] A template nucleic acid having homology with a target position in a gene or pathway described herein, e.g., in Section V B, e.g., in Table V - , VII- 14, VII- 15, VII- 16, VII- 17, VlI-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1, IX-IA, IX-2, IX-3, XIV-1, or Section VIII, can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.

[0580] The template nucleic acid can include sequence which, when integrated, results in: decreasing the activity of a positive control element; increasing the activity of a positive control element; decreasing the activity of a negative control element; increasing the activityof a negative control element; decreasing the expression of a gene; increasing the expression of a gene; increasing resistance to a disorder or disease; increasing resistance to viral entry; correcting a mutation or altering an unwanted amino acid residue conferring, increasing, abolishing or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule.

[0581] The template nucleic acid can include sequence which results in:

a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 or more nucleotides of the target sequence.

[0582] In an embodiment, the template nucleic acid is 20+/-10, 30+/-10, 40+/-10, 50+/-10, 60+/- 10, 70+/-10, 80+/-10, 90+/- 10, 100+/-10, 110+/-10, 120+/-10, 130+/-10, 140+/-10, 150+/-10, 160+/-10, 170+/-10, 180+/-10, 190+/-10, 200+/-10, 210+/-10, of 220+/-10 nucleotides in length.

[0583] In an embodiment, the template nucleic acid is 30+/-20, 40+/-20, 50+/-20, 60+/-20, 70+/- 20, 80+/-20, 90+/-20, 100+/-20, 110+/-20, 120+/-20, 130+/-20, 140+/-20, I50+/-20, 160+/-20, 170+/-20, 180+/-20, 190+/-20, 200+/-20, 210+/-20, of 220+/-20 nucleotides in length. [0584] In an embodiment, the template nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to300, 50 to 200, or 50 to 100 nucleotides in length.

[0585] A template nucleic acid comprises the following components:

[5' homology arm]-[replacement sequence]-[3' homology arm].

[0586] The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites.

[0587] In an embodiment, the 3' end of the 5' homology arm is the position next to the 5' end of the replacement sequence. In an embodiment, the 5' homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5' from the 5' end of the replacement sequence.

[0588] n an embodiment, the 5' end of the 3' homology arm is the position next to the 3' end of the replacement sequence. In an embodiment, the 3' homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3' from the 3' end of the replacement sequence.

[0589] It is contemplated herein that one or both homology arms may be shortened to avoid including certain sequence repeat elements, e.g., Alu repeats, LINE elements. For example, a 5' homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3' homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5' and the 3' homology arms may be shortened to avoid including certain sequence repeat elements.

[0590] It is contemplated herein that template nucleic acids for correcting a mutation may designed for use as a single-stranded oligonucleotide (ssODN). When using a ssODN, 5' and 3' homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made.

[0591] In an embodiment, an ssODN may be used to correct a mutation in a gene or pathway described herein, e.g., in Section V B, e.g., in Table VII- 1 , VII- 14, VII- 1 , VII- 16, VII- 17, Vll-1 8, VI1-19, VII-20, Vll-21, Vll-22, VII-23, VII-24, VII-25, IX- 1, 1X- 1A, IX-2, 1X-3, XlV-1, or Section VIII.

IV.2 NHEJ Approaches for Gene Targeting

[0592] As described herein, nuclease-induced non-homologous end-joining (NHEJ) can be used to target gene-specific knockouts. Nuclease-induced NHEJ can also be used to remove (e.g., delete) sequence in a gene of interest.

[0593] While not wishing to be bound by theory, it is believed that, in an embodiment, the genomic alterations associated with the methods described herein rely on nuclease-induced NHEJ and the error-prone nature of the NHEJ repair pathway. NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair. Two-thirds of these mutations typically alter the reading frame and, therefore, produce a non-functional protein. Additionally, mutations that maintain the reading frame, but which insert or delete a significant amount of sequence, can destroy functionality of the protein. This is locus dependent as mutations in critical functional domains are likely less tolerable than mutations in non-critical regions of the protein.

[0594] The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most . commonly in the 1-50 bp range, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells. [0595] Because EJ is a mutagenic process, it can also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. If a double-strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences; however, the error-prone nature of NHEJ may still produce indel mutations at the site of repair.

[0596] Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate NHEJ- mediated indels. NHEJ-mediated indels targeted to the gene, e.g., a coding region, e.g., an early coding region of a gene of interest can be used to knockout (i.e., eliminate expression of) a gene of interest. For example, early coding region of a gene of interest includes sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).

Placement of double strand or single strand breaks relative to the target position

[0597] In an embodiment, in which a gRNA and Cas9 nuclease generate a double strand break for the purpose of inducing NHEJ-mediated indels, a gRNA, e.g., a unimolecular (or chimeric) or modular gRNA molecule, is configured to position one double-strand break in close proximity to a nucleotide of the target position. In an embodiment, the cleavage site is between 0-500 bp away from the target position (e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position).

[0598] In an embodiment, in which two gRNAs complexing with Cas9 nickases induce two single strand breaks for the puipose of inducing NHEJ-mediated indels, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position two single-strand breaks to provide for NHEJ repair a nucleotide of the target position. In an embodiment, the gRNAs are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, essentially mimicking a double strand break. In an embodiment, the closer nick is between 0-30 bp away from the target position (e.g., less than 30, 25, 20, 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position), and the two nicks are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 0 bp). In an embodiment, the gRNAs are configured to place a single strand break on either side of a nucleotide of the target position.

[0599] Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate breaks both sides of a target position. Double strand or paired single strand breaks may be generated on both sides of a target position (e.g., of a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- , IX- 1A, ΓΧ -2, IX-3, XIV- 1, or Section VIII) to remove the nucleic acid sequence between the two cuts (e.g., the region between the two breaks is deleted). In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of a target position (e.g., the f st gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

IV.3 Targeted Knockdown

[0600] Unlike CRISPR/Cas-mediated gene knockout, which permanently eliminates expression by mutating the gene at the DNA level, CRISPR/Cas knockdown allows for temporary reduction of gene expression through the use of artificial transcription factors. Mutating key residues in both DNA cleavage domains of the Cas9 protein (e.g. the DIOA and H840A mutations) results in the generation of a catalytically inactive Cas9 (eiCas9 which is also known as dead Cas9 or dCas9). A catalytically inactive Cas9 complexes with a gRNA and localizes to the DNA sequence specified by that gRNA's targeting domain, however, it does not cleave the target DNA. Fusion of the dCas9 to an effector domain, e.g., a transcription repression domain, enables recruitment of the effector to any DNA site specified by the gRNA. While it has been show that the eiCas9 itself can block transcription when recruited to early regions in the coding sequence, more robust repression can be achieved by fusing a transcriptional repression domain (for example RA , SID or ERD) to the Cas9 and recruiting it to the promoter region of a gene. It is likely that targeting DNAsel hypersensitive regions of the promoter may yield more efficient gene repression or activation because these regions are more likely to be accessible to the Cas9 protein and are also more likely to harbor sites for endogenous transcription factors. Especially for gene repression, it is contemplated herein that blocking the binding site of an endogenous transcription factor would aid in downregulating gene expression. In another embodiment, an eiCas9 can be fused to a chromatin modifying protein. Altering chromatin status can result in decreased expression of the target gene.

[0601] In an embodiment, a gRNA molecule can be targeted to a known transcription response elements (e.g., promoters, enhancers, etc.), a known upstream activating sequences (UAS), and/or sequences of unknown or known function that are suspected of being able to control expression of the target DNA. [0602] CRISPR/Cas-mediated gene knockdown can be used to reduce expression of an unwanted allele or transcript. Contemplated herein are scenarios wherein permanent destruction of the gene is not ideal. In these scenarios, site-specific repression may be used to temporarily reduce or eliminate expression. It is also contemplated herein that the off-target effects of a Cas- repressor may be less severe than those of a Cas nuclease as a nuclease can cleave any DNA sequence and cause mutations whereas a Cas-repressor may only have an effect if it targets the promoter region of an actively transcribed gene. However, while nuclease-mediated knockout is pennanent, repression may only persist as long as the Cas-repressor is present in the cells. Once the repressor is no longer present, it is likely that endogenous transcription factors and gene regulatory elements would restore expression to its natural state.

IV.4 Examples of gRNAs in Genome Editing Methods

[0603] gRNA molecules as described herein can be used with Cas9 molecules that generate a double strand break or a single strand break to alter the sequence of a target nucleic acid, e.g., a target position or target genetic signature. gRNA molecules useful in these methods are described below.

[0604] In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;

a) it can position, e.g., when targeting a Cas9 molecule that makes double strand breaks, a double strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection; b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and c) (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 1, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain,

or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding

sequence of a naturally occurring S. pyogenes, S. thermophilics, S. aureus, or N.

meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides

from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no

more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally

occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain;

occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.

[0605] In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).

[0606] In an embodiment, the gRNA is configured such that it comprises properties: a and b(iij.

[0607] In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).

[0608] In an embodiment, the gRNA is configured such that it comprises properties: a and c .

[0609] In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c .

[0610] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i). [061 ] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).

[061 2] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).

[061 3] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).

[0 4] In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties; a) it can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection; b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and c) (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 1, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. tkermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than I , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. tkermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; (iii) there are at least 16, 1 , 21, 26, 3 , 32, 36, 4 1, 46, 50, 1, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 4 1, 46, 50, 51, or 54 nucleotides

121

i from the corresponding sequence of a naturally occurring S. pyogenes^ S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by

no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.

[0615] In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).

[0616] In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).

[0617] In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).

[0618] In an embodiment, the gRNA is configured such that it comprises properties: a and c.

[0619] In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.

[0620] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).

[0621] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).

[0622] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).

[0623] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).

[0624] In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation. [0625] In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

[0626] In an embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs, comprising a first and a second gRNA, is configured such that they comprises one or more of the following properties; a) one or both of the gRNAs can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break within (i) 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection; b) one or both have a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17 or, (ii) 18 nucleotides; c) for one or both: (i) the proximal and tail domain, when taken together, comprise at least 1 , 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain,

or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3'

to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N.

meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRN A, or a sequence that differs b

or, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or (v) the tail domain comprises 1 , 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; d) the gRNAs are configured such that, when hybridized to target nucleic acid, they are separated by 0-50, 0-100, 0-200, at least 10, at least 20, at least 30 or at least 50 nucleotides; e) the breaks made by the first gRNA and second gRNA are on different strands; and f) the PAMs are facing outwards.

[0627] In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(i).

[0628] In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ii).

[0629] In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iii).

[0630] In an embodiment, one or both of the gRNAs configured such that it comprises properties: a and c .

[0631] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a, b, and c .

[0632] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), and c(i).

[0633] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), and c(ii). [0634] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, and d.

[0635] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, and e .

[0636] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, d, and e.

[0637]In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), and c(i).

[0638] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), and c(ii).

[0639] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, and d.

[0640] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, and e.

[0641] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, d, and e.

[0642] In an embodiment, the gRNAs are used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the' RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D O, e.g., the DIOA mutation.

[0643] In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

V. Constructs/Components

[0644] The components, e.g., a Cas9 molecule or gRNA molecule, or both, can be delivered, formulated, or administered in a variety of forms, see, e.g., Table V-l a and Table V-lb. When a component is delivered encoded in DNA the DNA will typically include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 molecule sequences include CMV, EF- la, MSCV, PGK, CAG control promoters. Useful promoters for gRNAs include H1, EF- a and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment, a promoter for a Cas9 molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.

[0645] Table V-la and Table V-lb provide examples of how the components can be formulated, delivered, or administered.

Table V-la In an embodiment, the mRNA comprises one or more modifications, e.g., as described in Section X.

Protein DNA DNA In this embodiment a Cas9 molecule, typically an eaCas9 molecule, is provided as a protein. A gRNA is transcribed from DNA.

Protein RNA DNA In this embodiment an eaCas9 molecule is provided as a protein. A gRNA is provided as RNA. In an embodiment, the gRNA comprises one or more modifications, e.g., as described in Section X.

Table V-l b embodiment, the gRNA comprises one or more modifications, e.g., as described in Section X. In an embodiment, the mRNA comprises one or more modifications, e.g., as described in section X.

Protein DNA Yes In this embodiment a Cas9 molecule, typically an eiCas9 molecule, is provided as a protein. A gRNA is provided encoded in DNA.

Protein RNA Yes In this embodiment a Cas9 molecule, typically an eiCas9 molecule, is provided as a protein. A gRNA is provided as RNA. In an embodiment, the gRNA comprises one or more modifications, e.g., as described in Section X.

DNA-based Delivery of a Cas9 molecule and or a gRNA molecule

[0646] DNA encoding Cas9 molecules (e.g., eaCas9 molecules or eiCas9 molecules) and/or gRNA molecules, can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.

[0647] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).

[0648] A vector can comprise a sequence that encodes a Cas9 molecule and/or a gRNA molecule. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear local ization, nucleolar local ization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, a vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.

[0649] One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and a splice acceptor or donor can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter). In other embodiments, the promoter is recognized by RNA polymerase 111 (e.g., a U6 promoter). In some embodiments, the promoter is a regulated promoter (e.g., inducible promoter). In other embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a viral promoter. In other embodiments, the promoter is a non-viral promoter.

[0650] In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA vims). In other embodiments, the vims is an RNA vims (e.g., an ssRNA vims). Exemplary viral vectors/viruses include, e.g., retroviruses, lentivimses, adenovirus, adeno- associated virus (AAV), vaccinia vimses, poxviruses, and heipes simplex viruses.

[0651] In some embodiments, the vims infects dividing cells. In other embodiments, the vims infects non-dividing cells. In some embodiments, the vims infects both dividing and non- dividing cells. In some embodiments, the vims can integrate into the host genome. In some embodiments, the vims is engineered to have reduced immunity, e.g., in human. In some embodiments, the vims is replication-competent. In other embodiments, the vims is replication- defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In some embodiments, the virus causes transient expression of the Cas9 molecule and/or the gRN A molecule. In other embodiments, the vims causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRNA molecule. The packaging capacity of the vimses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 1 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

[0652] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant retrovirus. In some embodiments, the retrovirus (e.g., Moloney murine leukemia vims) comprises a reverse transcriptase, e.g., that allows integration into the host genome. In some embodiments, the retrovirus is replication-competent. In other embodiments, the retrovirus is replication-defective, e.g., having one of more coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted. [0653] n some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant lentivirus. For example, the lentivirus is replication-defective, e.g., does not comprise one or more genes required for viral replication.

[0654] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant adenovirus. In some embodiments, the adenovirus is engineered to have reduced immunity in human.

[0655] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant AAV. In some embodiments, the AAV can incorporate its genome into that of a host cell, e.g., a target cell as described herein. In some embodiments, the AAV is a self- complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods include, e.g., AAVl , AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y73 F and/or.T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8. AAV 8.2, AAV9, AAV rh 10, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods.

[0656] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a hybrid virus, e.g., a hybrid of one or more of the viruses described herein.

[0657] A Packaging cell is used to form a virus particle that is capable of infecting a host or target cell. Such a cell includes a 293 cell, which can package adenovirus, and a ψ2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into a host or target cell (if applicable), with other viral sequences being replaced by an expression cassette encoding the protein to be expressed. For example, an AAV vector used in gene therapy typically only possesses inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and gene expression in the host or target cell. The missing viral functions are supplied in trans by the packaging cell line. Henceforth, the viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0658] In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., geneticmodification of the viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibodie, a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).

[0659] In an embodiment, the viral vector achieves cell type specific expression. For example, a tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cell. The specificity of the vector can also be mediated by microRNA- dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of the viral vector and a target cell membrane. For example, a fusion protein such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, aviruse that requires the breakdown of the cell wall (during cell division) and therefore will not infect a non-diving cell can be altered to incorporate a nuclear localization peptide in the matrix protein of the virus thereby enabling the transduction of non-proliferating cells.

[0660] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a non- vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

[0661] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a combination of a vector and a non-vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer, e.g., in a respiratory epithelial cell than either a viral or a liposomal method alone.

[0662] In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non- viral vector is an inorganic nanoparticle (e.g., attached to the payload to the surface of the nanoparticle). Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe lvln0 ), or silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment

(e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.

[0663] Exemplary lipids for gene transfer are shown in Table XII-2.

[0664] Exemplary polymers for gene transfer are shown below in Table XII-3.

[0665] In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

[0666] In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the "empty" particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes - subject (i.e., patient) derived membrane-bound nanovescicle (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).

[0667] In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein. Delivery of RNA encoding a Cas9 molecule

[0668] RNA encoding Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof.

Delivery Cas9 molecule protein

[0669] Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) can be delivered into cells by art-known methods or as described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA.

Route of Administration

[0670] Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to the eye.

[0671] Local modes of administration include, by way of example, , intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen)), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum or substantia nigra intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intraparenchymal or intravitreal) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

[0672] n an embodiment, components described herein are delivered by intraparenchymal injection into discrete regions of the brain, including, e.g., regions comprising medium spiny neurons, or regions comprising cortical neurons. Injections may be made directly into more than one region of the brain.

[0673] In an embodiment, components described herein are delivered by subretinally, e.g., by subretinal injection. Subretinal injections may be made directly into the macular, e.g., submacular injection.

[0674] In an embodiment, components described herein are delivered by intravitreal injection. Intravitreal injection has a relatively low risk of retinal detachment risk. In an embodiment, a nanoparticle or viral vector, e.g., AAV vector, e.g., an AAV2 vector, e.g., a modified AAV2 vector, is delivered intravitreally.

[0675] In an embodiment, a nanoparticle or viral vector, e.g., AAV vector, delivery is via intraparenchymal injection.

[0676] Methods for administration of agents to the eye are known in the medical arts and can be used to administer components described herein. Exemplary methods include intraocular injection (e.g., retrobulbar, subretinal, submacular, intravitreal and intrachoridal), iontophoresis, eye drops, and intraocular implantation (e.g., intravitreal, sub-Tenons and sub-conjunctival).

[0677] Administration may be provided as a periodic bolus (for example, subretinally, intravenously or intravitreally) or as continuous infusion from an internal reservoir (for example, from an implant disposed at an intra- or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device immobilized to an inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT/USOO/00207, PCT/US02/14279, Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41: 181- 1185, and Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41 :1186-1 191). A variety of devices suitable for administering components locally to the inside of the eye are known in the art. See, for example, U.S. Pat. Nos. 6,25 1,090, 6,299,895, 6,416,777, 6,413,540, and PCT/USOO/28187.

[0678] n addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.

[0679] Representative synthetic, biodegradable polymers include, for example: polyamides such as poly( amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non- degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. [0680]Poly(lactide-co-glycolide) microsphere can also be used for intraocular injection. Typically the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein.

Bi-Modal or Differential Delivery of Components

[0681] Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.

[0682] In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, or template nucleic acid. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.

[0683] Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result- in more persistent expression of and presence of a component.

VI. PAY OA S

[0684] Cas9 molecules, typically eiCas9 molecules and gRNA molecules, e.g., an eiCas9 molecule/gRNA molecule complex, can be used to deliver a wide variety of payloads. In an embodiment, the payload is delivered to target nucleic acids or to chromatin, or other components, near or associated with a target nucleic acid.

[0685] While not wishing to be bound by theory, it is believed that the sequence specificity of the gRNA molecule of an eiCas9 molecule/gRNA molecule complex contributes to a specific interaction with the target sequence, thereby effecting the delivery of a payload associated with, e.g., covalently or noncovalently coupled to, the Cas9 molecule/gRNA molecule complex. [0686] In an embodiment, the payload is covalently or non-covalently coupled to a Cas9, e.g., an eiCas9 molecule. In an embodiment, the payload is covalently or non-covalently coupled to a gRNA molecule. In an embodiment, the payload is linked to a Cas9 molecule, or gRNA molecule, by a linker, e.g., a linker which comprises a bond cleavable under physiological conditions. In other embodiments the bond is not cleavable or is only poorly cleavable, under physiological conditions. In an embodiment, "covalently coupled" means as part of a fusion protein containing a Cas9 molecule.

Delivery of Multiple Payloads

[0687] In an embodiment, a first payload molecule is delivered by a first Cas9 molecule and a second payload molecule is delivered by a second Cas9 molecule. In an embodiment, the first and second payloads are the same. In an embodiment, first and second Cas9 molecules are the same, e.g. are from the same species, have the same PAM, and/or have the same sequence. In an embodiment, first and second Cas9 molecules are different, e.g. are from different species, have the different PAMs, and/or have different sequences. Examples of configurations are provided in Table VI- 1. Typically the Cas9 molecules of Table VI- 1 are eiCas9 molecules. In other embodiments a Cas9 molecule is selected such that payload delivery and cleavage are both effected. In an embodiment, multiple payloads, e.g., two payloads, is delivered with a single Cas9 molecule.

Table V - : Configurations for delivery of payloads by more than one Cas9 molecule/gRNA molecule complex second Cas9 molecule are guided by different gRNA molecules. C I C2 P I PI In this embodiment, the Cas9 molecules are different but each delivers the same payload. In an embodiment, the first and second Cas9 molecule are guided by different gRNA molecules. C I C2 PI P2 In this embodiment, the Cas9. molecules are different as are the payloads. In an embodiment, the first and second Cas9 molecule are guided by different gRNA molecules.

[0688] In an embodiment, two different drugs are delivered. In an embodiment, a first payload, e.g., a drug, coupled by a first linker to a first Cas9 molecule and a second payload, e.g., a drug, coupled by a second linker to a second Cas9 molecule are delivered. In an embodiment, the first and second payloads are the same, and, in an embodiment, are coupled to the respective Cas9 molecule by different linkers, e.g., having different release kinetics. In an embodiment, the first and second payloads are different, and, in an embodiment, are coupled to the respective Cas9 molecule by the same linker. In an embodiment, the first and second payload interact. E .g., the first and second payloads form a complex, e.g., a dimeric or multimeric complex, e.g., a dimeric protein. In an embodiment, the first payload can activate the second payload, e.g., the first payload can modify, e.g., cleave or phosphorylate, the second payload. In an embodiment the first payload interacts with the second payload to modify, e.g., increase or decrease, an activity of the second payload.

[0689] A payload can be delivered in vitro, ex vivo, or in vivo.

Classes of Payloads

[0690] A payload can comprise a large molecule or biologies (e.g., antibody molecules), a fusion protein, an amino acid sequence fused, as a fusion partner, to a Cas9 molecule, e.g., an eiCas9 molecule, an enzyme, a small molecules (e.g., HDAC and other chromatin modifiers/inhibitors, exon skipping molecules, transcription inhibitors), a microsatellite extension inhibitor, a carbohydrate, and DNA degraders (e.g., in an infectious disease or "foreign" DNA setting), a nucleic acid, e.g., a DNA, RNA, mRNA, siRNA, RNAi, or an antisense oligonucleotide.

[0691] Table VI-2 provides exemplary classes of payloads.

Table Vl-2

Exemplary Classes of Payloads Large Molecules Small Molecules Polymers Biologies Proteins and polypeptides, e.g., antibodies, enzymes, structural peptides, ligands, receptors, fusion proteins, fusion partners (as a fusion protein with a Cas9, e.g., and eiCas9) Carbohydrates HDAC and other chromatin modifiers/inhibitors Exon skipping molecules, Transcription inhibitors Microsatellite extension inhibitors Entities that degrade DNA

Large Molecules

[0692] In an embodiment a payload comprises a polymer, e.g., a biological polymer, e.g., a protein, nucleic acid, or carbohydrate.

[0693] In an embodiment the payload comprises a protein, biologic, or other large molecule (i.e., a molecule having a molecular weight of at least, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD). n an embodiment a payload comprises a polymer, e.g., a biological polymer, e.g., a protein, nucleic acid, or carbohydrate. The polymer can be a naturally occurring or non-naturally occurring polymer. In an embodiment, the payload is a natural product. For example, the natural product can be a large molecule or a small molecule. Polypeptides, Proteins

[0694] In an embodiment the payload comprises a protein or polypeptide, e.g., a protein or polypeptide covalently or non-covalently coupled to a Cas9 molecule.

[0695] In an embodiment, the protein or polypeptide is dimeric or multimeric, and each subunit is delivered by a Cas9 molecule. In an embodiment, a first protein and second protein are delivered by one or more Cas9 molecules, e.g., each by a separate Cas9 molecule or both by the same Cas9 molecule.

[0696] In an embodiment, the protein or polypeptide is linked to a Cas9 molecule by a linker, e.g., a linker which comprises a bond cleavable under physiological conditions. In an embodiment, a linker is a linker from Section XI herein. In an embodiment, the bond is not cleavable under physiological conditions.

Specific Binding Ligands, Antibodies

[0697] In an embodiment the payload comprises a ligand, e.g., a protein, having specific affinity for a counter ligand. In an embodiment, the ligand can be a receptor (or the ligand for a receptor), or an antibody.

[0698] In an embodiment a payload comprises an antibody molecule. Exemplary antibody molecules include, e.g., proteins or polypeptides that include at least one immunoglobulin variable domain. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al„ Eur J Immunol. 1996; 26(3):629-639)). For example, antigen-binding fragments of antibodies can include, e.g., (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and C I domains; (ii) a

F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See, e.g., US patents 5,260,203, 4,946,778, and 4,88 , 1 5; Bird et al. ( 88) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof)- Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred. In some embodiments, the antibody is a human antibody or humanized antibody.

[0699] In an embodiment, the antibody molecule is a single-domain antibody (e.g., an sdAb, e.g., a nanobody), e.g., an antibody fragment consisting of a single monomelic variable antibody domain. In an embodiment, the molecular weight of the single-domain antibody is about 12-15 kDa. For example, the single-domain antibody can be engineered from heavy-chain antibodies found in camelids (e.g., VHH fragments). Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG), e.g., from humans or mice, into monomers. Single-domain antibodies derived from either heavy or light chain can be obtained to bind specifically to target epitopes. For example, a single-domain antibody can be a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG.

[0700] Single-domain antibodies can have similar affinity to antigens as whole antibodies. They can also be more heat-resistant and/or stable towards detergents and high concentrations of urea. Those, e.g., derived from camelid and fish antibodies can be less lipophilic and more soluble in water, owing to their complementarity determining region 3 (CDR3), which forms an extended loop covering the lipophilic site that normally binds to a light chain. In an embodiment, the single-domain antibody does not show complement system triggered cytotoxicity, e.g., because they lack an Fc region. Single-domain antibodies, e.g., camelid and ish derived sdAbs, can bind to hidden antigens that may not be accessible to whole antibodies, for example to the active sites of enzymes. This property can result from their extended CDR3 loop, which is able to penetrate such sites.

[0701] A single-domain antibody can be obtained by immunization of, e.g., dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. By reverse transcription and polymerase chain reaction, a gene library of single-domain antibodies containing several million clones is produced. Screening techniques like phage display and ribosome display help to identify the clones binding the antigen.

[0702] A different method uses gene libraries from animals that have not been immunized beforehand. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step.

[0703] When the most potent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization is unproblematic because of the homology between, e.g., camelid VHH and human VH fragments. The final step is the translation of the optimized single-domain antibody in E. coli, Saccharomyces cerevisiae 'or other suitable organisms.

[0704] Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens. Monomerization is usually accomplished by replacing lipophilic by hydrophilic amino acids. If affinity can be retained, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other organisms.

[0705] In an embodiment, a payload comprises a transcription activator protein or domain, e.g., a VP 16 protein or domain, or a transcription repressor protein or domain. Fusion Proteins and Fusion Partners

[0706] n an embodiment the payload comprises a fusion protein. Exemplary fusion proteins include a first and second fusion partner, which can possess different functional properties or which can be derived from different proteins. In an embodiment, the fusion protein can comprise a first fusion partner that binds a nucleic acid and a second fusion partner that that comprises an enzymatic activity or that promotes or inhibits gene expression. n an embodiment, the payload itself is a fusion protein. In an embodiment, the payload is fused to a Cas9 molecule.

[0707] For example, the fusion protein can contain a segment that adds stability and/or deliverability to the fused protein. In some embodiments, the fusion protein can be a protein described herein (e.g., a receptor) fused to an immunoglobulin fragment (e.g., Fc fragment), transferring, or a plasma protein, e.g., albumin. The fusion protein can also contain a segment that adds toxicity to the fused protein (e.g. conveyed by toxins, enzymes or cytokines). Fusion proteins can also be used to enable delivery and/or targeting routes (e.g., by HIV-1 TAT protein). Other examples include, e.g., fusions that allow for mutivalency, such as streptavidin fusions, or fusions of two active components (e.g., with or without a cleavable linker in between).

[0708] In an embodiment, the protein or polypeptide is a fusion partner with a Cas9 molecule, e.g., an eiCas9 molecule.

[0709] In an embodiment, a payload comprises fusion partner with a Cas9 molecule comprising a transcription activator protein or domain, e.g., a VP16 protein or domain, or a transcription repressor protein or domain.

Enzymes

[0710] In an embodiment a payload comprises an enzyme. Exemplary enzymes include, e.g., oxidoreductases (e.g., catalyze oxidation/reduction reactions), transferases (e.g., transfer a functional group (e.g. a methyl or phosphate group)), hydrolases (e.g., catalyze the hydrolysis of various bonds), lyases (e.g., cleave various bonds by means other than hydrolysis and oxidation), isomerases (catalyze isomerization changes within a single molecule), and ligases (e.g., join two molecules with covalent bonds). In an embodiment an enzymes mediates or is associated with one or more functions in the cell nucleus, e.g., DNA synthesis, transcription, epigenetic modification of DNA and histones, RNA post-transcriptional modification, cell cycle control, DNA damage repair, or genomic instability.

Small Molecules

[0711] In an embodiment a payload comprises a small molecule compounds.

[0712] In an embodiment a small molecule is a regulator of a biological process. For example, a small molecule can bind to a second molecule, e.g., biopolymer, e.g., a carbohydrate, protein, polypeptide, or a nucleic acid, and in an embodiment, alter one or more of the structure, distribution, activity, or function of the second molecule. In some embodiments, the size of the small molecule is on the order of 10~9 m. In some embodiments, the molecular weight of the small molecule is, e.g., between 200 amu and 500 amu, between 300 amu and 700 amu, between 500 amu and 700 amu, between 700 amu and 900 amu, or between 500 amu and 900 amu.

[0713] Exemplary small molecules include histone deacetylase (HDAC) inhibitors (e.g., suberoylanilide hydroxamic acid (SAHA), or romidepsin), histone methyltransferase inhibitors (, DNA methyltransferase inhibitors (e.g., azacitidine (or 5-azacitidine), decitabine (or 5-aza-2'- deoxycytidine), or DNA replication inhibitors. Small molecules can also include, e.g., small nucleic acid molecules (1-4 bases depending upon the base, e.g., that would be under 2 kD) and peptides.

[0714] Exemplary classes of small molecules that may be used as payloads include, but are not limited to, 5-alpha Reductase Inhibitor, 5-alpha Reductase Inhibitors, 5-Lipoxygenase Inhibitor, 5-Lipoxygenase Inhibitors, Acetyl Aldehyde Dehydrogenase Inhibitors, Acetylcholine Release Inhibitor, Acetylcholine Release Inhibitors, Acetylcholine Releasing Agent, Acidifying Activity, Actinomycin, Actively Acquired Immunity, Adenosine Deaminase, Adenosine Receptor Agonist, Adenosine Receptor Agonists, Adenovirus Vaccines, Adrenal Steroid Synthesis Inhibitor, Adrenal Steroid Synthesis Inhibitors, Adrenergic Agonists, Adrenergic alpha-Agonists, Adrenergic alpha-Antagonists, Adrenergic alpha2-Agonists, Adrenergic beta-Agonists, Adrenergic beta-Antagonists, Adrenergic beta 1-Antagonists, Adrenergic beta2-Agonists, Adrenergic beta2-Antagonists, Adrenergic beta3-Agonists, Adrenergic Receptor Agonist, Adrenocorticotropic Hormone, Adrenocorticotropic Hormone, Aldehyde Dehydrogenase Inhibitor, Aldosterone Antagonist, Aldosterone Antagonists, Alkylating Activity, Alkylating Drug, Allergens, Allogeneic Cord Blood Hematopoietic Progenitor Cell Therapy, Allogeneic Cultured Cell Scaffold, Allylamine Antifungal, Allylamine, alpha Glucosidase Inhibitors, alpha- Adrenergic Agonist, alpha-Adrenergic Blocker, alpha-Glucosidase Inhibitor, alpha-Glucosidases, Aluminum Complex, Alveolar Surface Tension Reduction, Amide Local Anesthetic, Amides, Amino Acid Hypertonic Solution, Amino Acid, Amino Acids, Aminoglycoside Antibacterial, Aminoglycosides, Aminoketone, Aminosalicylate, Aminosalicylic Acids, Ammonium Ion Binding Activity, AMPA Receptor Antagonists, Amphenicol-class Antibacterial, Amphenicols, Amphetamine Anorectic, Amphetamines, Amylin Agonists, Amylin Analog, Androgen Receptor Agonists, Androgen Receptor Antagonists, Androgen Receptor Inhibitor, Androgen, Androstanes, Angiotensin 2 Receptor Antagonists, Angiotensin 2 Receptor Blocker, Angiotensin 2 Type 1Receptor Antagonists, Angiotensin Converting Enzyme Inhibitor, Angiotensin- converting Enzyme Inhibitors, Ant Venoms, Anthracycline Topoisomerase Inhibitor, Anthracyclines, Anti-anginal, Anti-coagulant, Anti-epileptic Agent, Anti-IgE, Anti-inhibitor Coagulant Complex, Antiarrhythmic, Antibodies, Monoclonal, Antibody-Surface Protein Interactions, Anticholinergic, Antidiarrheal, Antidiuretic Hormone Antagonists, Antidote for Acetaminophen Overdose, Antidote, Antiemetic, Antifibrinolytic Agent, Antigen Neutralization, Antigens, Bacterial, Antigens, Dermatophagoides, Antigens, Fungal, Antihelminthic, Antihistamine, Antimalarial, Antimetabolite Immunosuppressant, Antimetabolite, Antimycobacterial, Antiparasitic, Antiprotozoal, Antirheumatic Agent, Antiseptic, Antitoxins, Antivenin, Antivenins, Appetite Suppression, Aptamers, Nucleotide, Aromatase Inhibitor, Aromatase Inhibitors, Aromatic Amino Acid Decarboxylation Inhibitor, Arteriolar Vasodilation, Arteriolar Vasodilator, Asparaginase, Asparagine-specific Enzyme, Atypical Antipsychotic, Autologous Cellular Immunotherapy, Autologous Cultured Cell, Autonomic Ganglionic Blocker, Azole Antifungal, Azoles, B Lymphocyte Stimulator-directed Antibody Interactions, B Lymphocyte Stimulator-specific Inhibitor, Bacterial Neurotoxin Neutralization, Bacterial Proteins, Barbiturate, Barbiturates, BCG Vaccine, Bee Venoms, Benzodiazepine Antagonist, Benzodiazepine, Benzodiazepines, Benzothiazole, Benzothiazoles, Benzylamine Antifungal, Benzylamines, beta Lactamase Inhibitor, beta Lactamase Inhibitors, beta-Adrenergic Agonist, beta-Adrenergic Blocker, beta2-Adrenergic Agonist, beta3-Adrenergic Agonist, Biguanide, Biguanides, Bile Acid Sequestrant, Bile Acid, Bile Acids and Salts, Bile-acid Binding Activity, Bismuth, Bismuth, Bisphosphonate, Blood Coagulation Factor, Blood Coagulation Factors, Blood Viscosity Reducer, Bovine Intestinal Adenosine Deaminase, Bradykinin B2 Receptor Antagonist, Bradykinin B2 Receptor Antagonists, Calcineurin Inhibitor Immunosuppressant, Calcineurin Inhibitors, Calcitonin, Calcitonin, Calcium Channel Antagonists, Calcium Channel Blocker, Calcium Chelating Activity, Calcium, Calcium, Calcium-sensing Receptor Agonist, Calculi Dissolution Agent, Cannabinoid, Cannabinoids, Carbamoyl Phosphate Synthetase 1 Activator, Carbamoyl Phosphate Synthetase 1 Activators, Carbapenems, Carbon Radioisotopes, Carbonic Anhydrase Inhibitor, Carbonic Anhydrase Inhibitors, Cardiac Glycoside, Cardiac Glycosides, Carnitine Analog, Carnitine, Caseins, Catechol O-Methyltransferase Inhibitors, Catechol-O-Methyltransferase Inhibitor, Catecholamine Synthesis Inhibitor, Catecholamine Synthesis Inhibitors, Catecholamine, Catecholamine-depleting Sympatholytic, Catecholamines, Cations, Divalent, CCR5 Co-receptor Antagonist, CD20-directed Antibody Interactions, CD20- directed Cytolytic Antibody, CD20-directed Radiotherapeutic Antibody, CD25-directed Cytotoxin, CD3 Blocker Immunosuppressant, CD3 Receptor Antagonists, CD3-directed Antibody Interactions, CD30-directed Antibody Interactions, CD30-directed Immunoconjugate, CD52-directed Antibody Interactions, CD52-directed Cytolytic Antibody, CD80-directed Antibody Interactions, CD86-directed Antibody Interactions, Cell Death Inducer, Cell-mediated Immunity, Cells, Allogeneic, Cells, Cultured, Allogeneic, Cells, Cultured, Autologous, Cells, Epidermal, Central alpha-2 Adrenergic Agonist, Central Nervous System Depressant, Central Nervous System Depression, Central Nervous System Stimulant, Central Nervous System Stimulation, Centrally-mediated Muscle Relaxation, Cephalosporin Antibacterial, Cephalosporins, Chemokine Co-receptor 5 Antagonists, Chloride Channel Activation Potentiators, Chloride Channel Activator, Chloride Channel Activators, Cholecalciferol, Cholecystokinin Analog, Cholecystokinin, Cholinergic Agonists, Cholinergic Antagonists, Cholinergic Muscarinic Agonist, Cholinergic Muscarinic Agonists, Cholinergic Muscarinic Antagonist, Cholinergic Muscarinic Antagonists, Cholinergic Nicotinic Agonist, Cholinergic Receptor.Agonist, Cholinesterase Inhibitor, Cholinesterase Inhibitors, Cholinesterase Reactivator, Cholinesterase Reactivators, Chondrocytes, Collagen, Collagen-specific Enzyme, Collagenases, Competitive Opioid Antagonists, Complement Inhibitor, Complement Inhibitors, Contrast Agent for Ultrasound Imaging, Copper Absoiption Inhibitor, Copper, Copper- containing Intrauterine Device, Corticosteroid Hormone Receptor Agonists, Corticosteroid, CTLA-4-directed Antibody Interactions, CTLA-4-directed Blocking Antibody, Cyclooxygenase Inhibitors, Cysteine Depleting Agent, Cystic Fibrosis Transmembrane Conductance Regulator Potentiator, Cystine Disulfide Reduction, Cytochrome P450 1A2 Inhibitors, Cytochrome P450 2B6-Inducers, Cytochrome P450 2C19 Inducers, Cytochrome P450 2C 1 Inhibitors, Cytochrome P450 2C8 Inducers, Cytochrome P450 2C8 Inhibitors, Cytochrome P450 2C9 Inducers, Cytochrome P450 2C9 Inhibitors, Cytochrome P450 2D6 Inhibitor, Cytochrome P450 2D6 Inhibitors, Cytochrome P450 3A Inducers, Cytochrome P450 3A Inhibitors, Cytochrome P450 3A4 Inducers, Cytochrome P450 3A4 Inhibitors, Cytochrome P450 3A5 Inhibitors, Cytomegalovirus Nucleoside Analog DNA Polymerase Inhibitor, Cytoprotective Agent, Dander, Decarboxylase Inhibitor, Decarboxylase Inhibitors, Decreased Autonomic Ganglionic Activity, Decreased B Lymphocyte Activation, Decreased Blood Pressure, Decreased Cell Wall Integrity, Decreased Cell Wall Synthesis & Repair, Decreased Central Nervous System Disorganized Electrical Activity, Decreased Central Nervous System Organized Electrical Activity, Decreased Cholesterol Absorption, Decreased Coagulation Factor Activity, Decreased Copper Ion Absorption, Decreased Cytokine Activity, Decreased DNA Replication, Decreased Embryonic Implantation, Decreased Fibrinolysis, Decreased GnRH Secretion, Decreased Histamine Release, Decreased IgE Activity, Decreased Immunologic Activity, Decreased Immunologically Active Molecule Activity, Decreased Leukotriene Production, Decreased Mitosis, Decreased Parasympathetic Acetylcholine Activity, Decreased Platelet Aggregation, Decreased Platelet Production, Decreased Prostaglandin Production, Decreased Protein Synthesis, Decreased Renal + Excretion, Decreased Respiratory Secretion Viscosity, Decreased RNA Replication, Decreased Sebaceous Gland Activity, Decreased Sperm Motility, Decreased Striated Muscle Contraction, Decreased Striated Muscle Tone, Decreased Sympathetic Activity, Decreased Tracheobronchial Stretch Receptor Activity, Decreased Vascular Permeability, Demulcent

Activity, Demulcent, Deoxyribonuclease I, Deoxyuridine, Depigmenting Activity, Depigmenting Agent, Depolarizing Neuromuscular Blocker, Diagnostic Dye, Dietary Cholesterol Absorption Inhibitor, Dietary Proteins, Digestive/GI System Activity Alteration, Digoxin Binding Activity, Dihydrofolate Reductase Inhibitor Antibacterial, Dihydrofolate Reductase Inhibitor Antimalarial, Dihydrofolate Reductase Inhibitors, Dihydroorotate Dehydrogenase Inhibitors, Dihydropyridine Calcium Channel Blocker, Dihydropyridines, Dipeptidase Inhibitors, Dipeptidyl Peptidase 4 Inhibitor, Dipeptidyl Peptidase 4 Inhibitors, Diphosphonates, Diphtheria Toxin, Direct Thrombin Inhibitor, DNA Polymerase Inhibitors, DOPA Decarboxylase Inhibitors, Dopamine Agonists, Dopamine D2 Antagonists, Dopamine Uptake Inhibitors, Dopamine-2 Receptor Antagonist, Dopaminergic Agonist, Dyes, Echinocandin Antifungal, Egg Proteins, Dietary, Emesis Suppression, Endogenous Antigen Neutralization, Endoglycosidase, Endothelin Receptor Antagonist, Endothelin Receptor Antagonists, Enzyme Precursors, Epidermal Growth Factor Receptor Antagonist, Ergocalciferols, Ergolines, Ergot Alkaloids, Ergot Derivative, Ergot- derived Dopamine Receptor Agonist, Ergotamine Derivative, Ergotamines, Erythropoiesis- stimulating Agent, Erythropoietin, Ester Local Anesthetic, Esters, Estradiol Congeners, Estradiol, Estrogen Agonist/Antagonist, Estrogen Receptor Agonists, Estrogen Receptor Antagonist, Estrogen Receptor Antagonists, Estrogen, Estrogens, Conjugated (USP), Factor VIII

Activator, Factor VIII, Factor Xa Inhibitor, Factor Xa Inhibitors, Fatty Acids, Omega-3,

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Agonists, Gonadotropin Releasing Hormone Receptor Antagonists, Gonadotropin, Gonadotropins, Grain Proteins, Granulocyte Colony- Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, Growth Hormone Receptor Antagonist, Growth Hormone Receptor Antagonists, Growth Hormone Releasing Factor Analog, Guanylate Cyclase Activators, Guanylate Cyclase Stimulators, Guanylate Cyclase-C Agonist, HEALTHCARE/PHARMACEUTICAL INDUSTRY MENU, Home, News, DailyMed Announcements, Get RSS News & Updates, Search, Advanced Search, Browse Drug Classes, Labels Archives, Tablet/Capsule ID Tool, FDA Guidances & Information, NLM SPL Resources, Download Data, All Drug Labels, All Index Files, All Mapping Files, SPL Image Guidelines, Presentations & Articles, Application Development Support, Resources, Web Services, Mapping Files, Help, SWITCH TO CONSUMER/PATIENT MENU, HCV NS3/4A Protease Inhibitors, Hedgehog Pathway Inhibitor, Helicobacter pylori Diagnostic, Hematologic Activity Alteration, 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Inhibitor, Human Immunodeficiency Virus 1Fusion Inhibitor, Human Immunodeficiency Virus 1 Non-Nucleoside Analog Reverse Transcriptase Inhibitor, Human Immunodeficiency Virus Integrase Strand Transfer Inhibitor, Human Immunodeficiency Virus Nucleoside Analog Reverse Transcriptase Inhibitor, Human Immunoglobulin G, Human Immunoglobulin, Human Platelet-derived Growth Factor, Human Serum Albumin, Hydrolytic Lysosomal Glucocerebroside-specific Enzyme, Hydrolytic Lysosomal Glycogen-specific Enzyme, Hydrolytic Lysosomal Glycosaminoglycan-specific Enzyme, Hydrolytic Lysosomal Neutral Glycosphingolipid-specific Enzyme, Hydroxymethylglutaryl-CoA Reductase Inhibitors, Hydroxyphenyl-Pyruvate Dioxygenase Inhibitor, Hydroxyphenylpyruvate Dioxygenase Inhibitors, IgE-directed Antibody Interactions, Immunoconjugates, Immunoglobulin G, Immunoglobulins, Inactivated Salmonella Typhi Vaccine, Increased Acetylcholine Activity, Increased Blood Pressure, Increased Calcium-sensing Receptor Sensitivity, Increased Cellular Death, Increased Coagulation Activity, Increased Coagulation Factor Activity, Increased Coagulation Factor IX Activity, Increased Coagulation Factor VIII Activity, Increased Coagulation Factor VIII Concentration, Increased Coagulation Factor X Activity, Increased Cytokine Activity, Increased Cytokine Production, Increased Diuresis at Loop of Henle, Increased Diuresis, Increased Dopamine Activity, Increased Epithelial Proliferation, Increased Erythroid Cell Production, Increased Fibrin Polymerization Activity, Increased GHRH Activity, Increased Glutathione Concentration, Increased Hematopoietic Stem Cell Mobilization, Increased Histamine Release, Increased IgG Production, Increased Immunologically Active Molecule Activity, Increased Intravascular Volume, Increased Large Intestinal Motility, Increased Lymphocyte Activation, Increased Lymphocyte Cell Production, Increased Macrophage Proliferation, Increased Medullary Respiratory Drive, Increased Megakaryocyte Maturation, Increased Myeloid Cell Production, Increased Norepinephrine Activity, Increased Oncotic Pressure, Increased Platelet Aggregation, Increased Platelet Production, Increased Prostaglandin Activity, Increased Prothrombin Activity, Increased Sympathetic Activity, Increased T Lymphocyte Activation, Increased T Lymphocyte Destruction, Increased Thrombolysis, Increased Uterine Smooth Muscle Contraction or Tone, Influenza A M2 Protein Inhibitor, Inhalation Diagnostic Agent, Inhibit Ovum Fertilization, Insect Proteins, Insulin Analog, Insulin, Insulin, Integrin Receptor Antagonist, Integrin Receptor Antagonists, Interferon Alfa-2a, Interferon Alfa-2b, Interferon alpha, Interferon gamma, Interferon Inducers, Interferon-alpha, Interferon-beta, Interferon-gamma, Interleukin 1 Receptor Antagonists, Interleukin 2 Receptor Antagonists, Interleukin 2 Receptor-directed Antibody Interactions, Interleukin 6 Receptor Antagonists, Interleukin- 1 Receptor Antagonist, Interleukin- 2 Receptor Blocking Antibody, Interleukin-2, Interleukin-6 Receptor Antagonist, Intestinal Lipase Inhibitor, Iodine, Iron Chelating Activity, Iron Chelator, Iron, Irrigation, Kallikrein

Inhibitors, Keratinocytes, Ketolide Antibacterial, Ketolides, Kinase Inhibitor, 1-Thyroxine, 1- Triiodothyronine, Lead Chelating Activity, Lead Chelator, Leukocyte Growth Factor, Leukotriene Receptor Antagonist, Leukotriene Receptor Antagonists, Lincosamide Antibacterial, Lincosamides, Lipase Inhibitors, Lipid-based Polyene Antifungal, Lipopeptide Antibacterial, Lipopeptides, Live Attenuated Bacillus Calmette-Guerin Immunotherapy, Live Attenuated Bacillus Calmette-Guerin Vaccine, Live Attenuated Mumps Virus Vaccine, Live Human Adenovirus Type 4 Vaccine, Live Human Adenovirus Type 7 Vaccine, Live Rotavirus Vaccine, Local Anesthesia, Local Anesthetic, Loop Diuretic, Low Molecular Weight Heparin, Lymphocyte Function Alteration, Lymphocyte Growth Factor, M2 Protein Inhibitors, Macrolide Antibacterial, Macrolide Antimicrobial, Macrolide, Macrolides, Magnesium Ion Exchange Activity, Magnetic Resonance Contrast Activity, Mast Cell Stabilizer, Meat Proteins, Megakaryocyte Growth Factor, Melanin Synthesis Inhibitor, Melanin Synthesis Inhibitors, Melatonin Receptor Agonist, Melatonin Receptor Agonists, Metal Chelating Activity, Metal Chelator, Methylated Sulfonamide Antibacterial, Methylated Sulfonamides, Methylating Activity, Methylating Agent, Methylxanthine, Microtubule Inhibition, Microtubule Inhibitor, Milk Proteins, Monoamine Oxidase Inhibitor, Monoamine Oxidase Inhibitors, Monoamine Oxidase Type B Inhibitor, Monoamine Oxidase-B Inhibitors, Monobactam Antibacterial, Monobactams, Mood Stabilizer, TOR Inhibitor Immunosuppressant, TOR Inhibitors, Mucocutaneous Epithelial Cell Growth Factor, Mucolytic, Mumps Vaccine, Muscle Relaxant, N- Calcium Channel Receptor Antagonists, N-methyl-D-aspartate Receptor Antagonist, N- substituted Glycines, N-type Calcium Channel Antagonist, Natriuretic Peptide, Natriuretic Peptides, Neuraminidase Inhibitor, Neuraminidase Inhibitors, Neurokinin 1 Antagonists, Neuromuscular Depolarizing Blockade, Neuromuscular Nondepolarizing Blockade, Nicotine, Nicotinic Acid, Nicotinic Acids, Nitrate Vasodilator, Nitrates, Nitrofuran Antibacterial, Nitrofurans, Nitrogen Binding Agent, Nitrogen Mustard Compounds, Nitroimidazole Antimicrobial, Nitroimidazoles, NMDA Receptor Antagonists, Non-narcotic Antitussive, Non- Nucleoside Analog, Non-Nucleoside Reverse Transcriptase Inhibitors, Non-Standardized Animal Dander Allergenic Extract, Non-Standardized Animal Hair Allergenic Extract, Non- Standardized Animal Skin Allergenic Extract, Non-Standardized Bacterial Allergenic Extract, Non-Standardized Chemical Allergen, Non-Standardized Feather Allergenic Extract, Non- Standardized Food Allergenic Extract, Non-Standardized Fungal Allergenic Extract, Non- Standardized House Dust Allergenic Extract, Non-Standardized Insect Allergenic Extract, Non- Standardized Insect Venom Allergenic Extract, Non-Standardized Plant Allergenic Extract, Non- Standardized Plant Fiber Allergenic Extract, Non-Standardized Pollen Allergenic Extract, Noncompetitive AMPA Glutamate Receptor Antagonist, Nondepolarizing Neuromuscular Blocker, Nonergot Dopamine Agonist, Nonsteroidal Anti-inflammatory Compounds, Nonsteroidal Anti-inflammatory Drug, Norepinephrine Reuptake Inhibitor, Norepinephrine Uptake Inhibitors, Nucleic Acid Synthesis Inhibitors, Nucleoside Analog Antifungal, Nucleoside Analog Antiviral, Nucleoside Analog, Nucleoside Metabolic Inhibitor, Nucleoside Reverse Transcriptase Inhibitors, Nut Proteins, Oligonucleotides, Omega-3 Fatty Acid, Opioid Agonist, Opioid Agonist/Antagonist, Opioid Agonists, Opioid Antagonist, Opioid Antagonists, Organic Anion Transporting Polypeptide 1B 1 Inhibitors, Organic Anion Transporting Polypeptide 1B3 Inhibitors, Organic Anion Transporting Polypeptide 2B1 Inhibitors, Organic Cation Transporter 2 Inhibitors, Organometallic Compounds, Osmotic Activity, Osmotic Diuretic, Osmotic Laxative, Oxazolidinone Antibacterial, Oxazolidinones, Oxytocic, Oxytocin, P-Glycoprotein Inhibitors, P-Glycoprotein Interactions, P2Y12 Platelet Inhibitor, P2Y12 Receptor Antagonists, Paramagnetic Contrast Agent, Parathyroid Hormone Analog, Parathyroid Hormone, Parenteral Iron Replacement, Partial Cholinergic Nicotinic Agonist, Partial Cholinergic Nicotinic Agonists, Partial Opioid Agonist, Partial Opioid Agonist/Antagonist, Partial Opioid Agonists, Passively Acquired Immunity, Pediculicide, peginterferon alfa-2a, peginterferon alfa-2b, Penem Antibacterial, Penicillin-class Antibacterial, Penicillins, Peripheral Blood Mononuclear Cells, Peroxisome Prolif'erator Receptor alpha Agonist, Peroxisome Proliferator Receptor gamma Agonist, Peroxisome Proliferator-activated Receptor Activity, Peroxisome Proliferator-activated Receptor alpha Agonists, Phenothiazine, Phenothiazines, Phenylalanine Hydroxylase Activator, Phenylalanine Hydroxylase Activators, Phosphate Binder, Phosphate Chelating Activity, Phosphodiesterase 3 Inhibitor, Phosphodiesterase 3 Inhibitors, Phosphodiesterase 5 Inhibitor, Phosphodiesterase 5 Inhibitors, Photoabsorption, Photoactivated Radical Generator, Photoenhancer, Photosensitizing Activity, Plant Proteins, Plasma Volume Expander, Platelet Aggregation Inhibitor, Platelet-Derived Growth Factor, Platelet-reducing Agent, Platinum-based Drug, Platinum-containing Compounds, Pleuromutilin Antibacterial, pleuromutilin, Pollen, Polyene Antifungal, Polyene Antimicrobial, Polyenes, Polymyxin-class Antibacterial, Polymyxins, Porphyrin Precursor, Porphyrinogens, Positron Emitting Activity, Potassium Channel Antagonists, Potassium Channel Opener, Potassium Channel Openers, Potassium Compounds, Potassium Salt, Potassium-sparing Diuretic, Poultry Proteins, PPAR alpha, PPAR gamma, Progestational Hormone Receptor Antagonists, Progesterone Congeners, Progesterone, Progesterone, Progestin Antagonist, Progestin, Progestin-containing Intrauterine Device, Prostacycline Vasodilator, Prostacycline, Prostaglandin Analog, Prostaglandin El Agonist, Prostaglandin E l Analog, Prostaglandin Receptor Agonists, Prostaglandins E, Synthetic,

Prostaglandins I, Prostaglandins, Protease Inhibitor, Proteasome Inhibitor, Proteasome Inhibitors, Protein C, Protein Kinase Inhibitors, Protein Synthesis Inhibitors, Proton Pump Inhibitor, Proton Pump Inhibitors, Provitamin D2 Compound, Psoralen, Psoralens, Purine Antimetabolite, Purines, Pyrethrins, Pyrethroid, Pyrimidine Synthesis Inhibitor, Pyrophosphate Analog DNA Polymerase Inhibitor, Pyrophosphate Analog, Quaternary Ammonium Compounds, Quinolone Antimicrobial, Quinolones, Radioactive Diagnostic Agent, Radioactive Therapeutic Agent, Radioactive Tracers, Radiographic Contrast Agent, Radiopharmaceutical Activity, RANK Ligand Blocking Activity, RANK Ligand Inhibitor, Receptor Tyrosine Kinase Inhibitors, Recombinant Antithrombin, Recombinant Fusion Proteins, Recombinant Human

Deoxyribonuclease 1, Recombinant Human Growth Hormone, Recombinant Human Growth Hormones, Recombinant Human Interferon beta, Recombinant Proteins, Reducing and Complexing Thiol, Reduction Activity, Renal Dehydropeptidase Inhibitor, Renin Inhibitor, Renin Inhibitors, Respiratory Stimulant, Respiratory Syncytial Virus Anti-F Protein Monoclonal Antibody, Retinoid, Retinoids, Reversed Anticoagulation Activity, Rifamycin Antibacterial, Rifamycin Antimycobacterial, Rifamycins, RNA Synthetase Inhibitor Antibacterial, RNA Synthetase Inhibitors, Rotavirus Vaccines, Salivary Proteins and Peptides, Sclerosing Activity, Sclerosing Agent, Seed Storage Proteins, Selective Estrogen Receptor Modulators, Selective T Cell Costimulation Blocker, Selective T Cell Costimulation Modulator, Serotonin l b Receptor Agonists, Serotonin Id Receptor Agonists, Serotonin 2c Receptor Agonists, Serotonin 3 Receptor Antagonists, Serotonin 4 Receptor Antagonists, Serotonin and Norepinephrine Reuptake Inhibitor, Serotonin Reuptake Inhibitor, Serotonin Uptake Inhibitors, Serotonin- b and Serotonin- Id Receptor Agonist, Serotonin-2c Receptor Agonist, Serotonin-3 Receptor Antagonist, Serotonin-4 Receptor Antagonist, Serum Albumin, Shellfish Proteins, Sigma- 1 Agonist, Sigma-1 Receptor Agonists, Silk, Skeletal Muscle Relaxant, Skin Barrier Activity, Skin Test Antigen, Smoothened Receptor Antagonists, Sodium-Glucose Cotransporter 2 Inhibitor, Sodium-Glucose Transporter 2 Inhibitors, Soluble Guanylate Cyclase Stimulator, Somatostatin Analog, Somatostatin Receptor Agonists, Sphingosine 1-phosphate Receptor Modulator, Sphingosine 1-Phosphate Receptor Modulators, Standardized Animal Hair Allergenic Extract, Standardized Animal Skin Allergenic Extract, Standardized Chemical Allergen, Standardized Insect Allergenic Extract, Standardized Insect Venom Allergenic Extract, Standardized Pollen Allergenic Extract, Starch, Stimulant Laxative, Stimulation Large Intestine Fluid/Electrolyte Secretion, Streptogramin- Antibacterial, Streptogramins, Substance P/Neurokinin-1 Receptor Antagonist, Sucrose-specific Enzyme, Sulfonamide Antibacterial, Sulfonamide Antimicrobial, Sulfonamides, Sulfone, Sulfones, Sulfonylurea Compounds, Sulfonylurea, Surfactant Activity, Surfactant, Sympathomimetic Amine Anorectic, Sympathomimetic-like Agent, T Lymphocyte Costimulation Activity Blockade, Tetracycline-class Antibacterial, Tetracycline-class Antimicrobial, Tetracycline-class Drug, Tetracyclines, Thalidomide Analog, Thiazide Diuretic, Thiazide-like Diuretic, Thiazides, Thiazolidinedione, Thiazolidinediones, Thrombin Inhibitors, Thrombolytic Agent, Thrombopoiesis Stimulating Agent, Thrombopoietin Receptor Agonists, Thrombopoietin Receptor Interactions, Thrombopoietin, Thyroid Hormone Synthesis Inhibitor, Thyroid Hormone Synthesis Inhibitors, Thyroid Stimulating Hormone, Thyrotropin, Thyroxine, Tissue Scaffolds, Topoisomerase Inhibitor, Topoisomerase Inhibitors, Transglutaminases, Tricyclic Antidepressant, Triiodothyronine, Trypsin Inhibitors, Tuberculosis Skin Test, Tumor Necrosis Factor alpha Receptor Blocking Activity, Tumor Necrosis Factor Blocker, Tumor Necrosis Factor Receptor Blocking Activity, Typical Antipsychotic, Ultrasound Contrast Activity, Uncompetitive N-methyl-D-aspartate Receptor Antagonist, Uncompetitive NMDA Receptor Antagonists, Unfractionated Heparin, Urate Oxidase, Urea, Urease Inhibitor, Urease Inhibitors, Uric Acid-specific Enzyme, Vaccines, Attenuated, Vaccines, Inactivated, Vaccines, Live, Unattenuated, Vaccines, Typhoid, Vascular Endothelial Growth Factor Receptor Inhibitors, Vascular Endothelial Growth Factor-directed Antibody Interactions, Vascular Endothelial Growth Factor-directed Antibody, Vascular Sclerosing Activity, Vasodilation, Vasodilator, Vasopressin Analog, Vasopressin Antagonist, Vasopressins, Vegetable Proteins, Venom Neutralization, Venous Vasodilation, Vi polysaccharide vaccine, typhoid, Vinca Alkaloid, Vinca Alkaloids, Virus Neutralization, Virus-specific Hyperimmune Globulins, Vitamin A, Vitamin A, Vitamin B 12, Vitamin B Complex Compounds, Vitamin B Complex Member, Vitamin B12, Vitamin D Analog, Vitamin D, Vitamin D, Vitamin D2 Analog, Vitamin D3 Analog, Vitamin K Antagonist, Vitamin K Inhibitors, Vitamin K, Vitamin K, von Willebrand Factor, Warfarin Reversal Agent, Wasp Venoms, X-Ray Contrast Activity, Xanthine Oxidase Inhibitor, Xanthine Oxidase Inhibitors, Xanthines, or combinations thereof.

Microsatellitc extension inhibitors

[0715] In an embodiment a payload comprises a microsatellite extension inhibitor. In an embodiment, the microsatellite extension inhibitor is a DNA mismatch repair protein. Exemplary DNA mismatch repair proteins that can be delivered by the molecules and methods described herein include, e.g., MSH2, MSH3, MSH6, MLH1 , MLH3, PMS1, PMS2. Signal generators, radionuclides, reporter molecules, diagnostic probes

[071 ] In an embodiment a payload comprises a molecule that generates a signal. Such payloads are useful, e.g., in research, therapeutic (e.g., cancer therapy) and diagnostic applications. In an embodiment, the signal comprises: an electromagnetic emission, e.g., in the infrared, visible, or ultraviolet range; a particle, e.g., a product of radioactive decay, e.g., an alpha, beta, or gamma particle; a detectable substrate, e.g., a colored substrate; a reaction product, e.g., the product of an enzymatic reaction; or a ligand detectable by a specific binding agent, e.g., an antibody; or a dye. In an embodiment the signal comprises a fluorescent emission, e.g., by a fluorescent protein. Exemplary fluorescent proteins include, B ue UV Proteins (e.g., TagBFP, mTagBFP, Azurite, EBFP2, mKalamal, Si ius, Sapphire, T-Sapphire), Cyan Proteins (e.g., ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl), Green Proteins (e.g., EGFP, Emerald, Supeifolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen), Yellow Proteins (e.g., EYFP, Citrine, Venus, SYFP2,

TagYFP), Orange Proteins (e.g., Monomeric Kusabira-Orange, Ώ ΚΟΚ , mK02, mOrange, mOrange2), Red Proteins (mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2), Far-Red Proteins (e.g., mPlum, HcRed- Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP), Long Stokes Shift Proteins (e.g., mKeima Red, LSS-mKatel, LSS-mKate2, mBeRFP), Photoactivatible Proteins (e.g., PA- GFP, PAmCherryl, PATagRFP), Photoconvertible Proteins (e.g., Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange), Photoswitchable Proteins (e.g., Dronpa).

[0717] In an embodiment, a signal producing moiety is provided as the fusion partner of a Cas9 molecule, e.g., an eiCas9 molecule.

[0718] Signal generators or reporters, useful, e.g., for labelingr polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., indium ( In), iodine ( 1 1 or 1 I), yttrium ( 0Y), lutetium ( 7 Lu), actinium ( Ac), bismuth (2 Bi or 2 Bi), sulfur ( S), carbon ( C), tritium ( H), rhodium ( Rh), technetium ( mTc), praseodymium, or phosphorous ( ~P) or a positron-emitting radionuclide, e.g., carbon- 11 ("C), potassium-40 ( 0K), nitrogen- 13 (1 N), oxygen-15 ( 50), fluorine-18 ( F), and iodine-121 ( 11)), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups (which can be detected by a marked avidin, e.g., a molecule containing a streptavidin moiety and a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[07 ] In an embodiment, a payload comprises a radionuclide. The radionuclide can be incorporated into the gRNA molecule, the Cas9 molecule, or into a payload molecule. Exemplary radionuclides include, e.g., beta emitters, alpha emitters or gamma emitters. In an embodiment the radionuclide is iodine, e.g., 1I or 12 I, yttrium, e.g., 0Y, lutetium, e.g., 7 Lu, Actinium, e.g., Ac, bismuth, e.g., Bi or Bi), sulfur, e.g., S), carbon, e.g., C, tritium, H), rhodium, e.g., 1 Rh, technetium, e.g., Tc, praseodymium, or phosphorous, e.g., P.

Modulators of DNA and Chromatin Structure

[0720] In an embodiment a payload comprises an endogenous or exogenous modulator of DNA structure. A modulator, as is typical of payloads, can be delivered in vitro, ex vivo, or in vivo.

[0721] n an embodiment, the payload comprises a modulator of an epigenetic state or characteristic of DNA. In an embodiment an epigenetic state or characteristic can be altered to treat a disorder, or to influence the developmental or other state of a cell.

[0722] In an embodiment, the epigenetic state or characteristic comprises DNA methylation. For example, the payloads described herein can modulate the addition of methyl groups to DNA, e.g., to convert cytosine to 5-methylcytosine, e.g., at CpG sites.

[0723] Aberrant DNA methylation patterns (e.g., hypermethylation and hypomethylation compared to normal tissue) are associated with various diseases and conditions, e.g., cancer. The modulators described herein can be used to reactivate transcriptionally silenced genes or to inhibit transcriptionally hyperactive genes, e.g., to treat diseases, e.g., cancer.

[0724] DNA methylation can affect gene transcription. Genes with high levels of 5- methylcytosine, e.g., in their promoter region, can be transcriptionally less active or silent. Thus, methods described herein can be used to target and suppress transcriptional activity, e.g., of genes described herein.

[0725] In some embodiments, the modulator promotes maintenance of D A methylation. For example, the modulators can have DNA methyltransferase (DNMT) activity or modulate DNMT activity, e.g\, to maintain DNA methylation or reduce passive DNA demethylation, e.g., after DNA replication.

[0726] In some embodiments, the modulator promotes de novo DNA methylation. For example, the modulators described herein can have de novo DNA methyltransferase (DNMT) (e.g., DNMT3a, DNMT3b, DNMT3L) activity or modulate de novo DNMT (e.g., DNMT3a, DNMT3b, DNMT3L) activity, e.g., to produce DNA methylation patterns, e.g., early in development.

[0727] Epigenetic changes in DNA (e.g., methylation), can be evaluated by art-known methods or as described herein. Exemplary methods for detecting DNA methylation include, e.g., Methylation-Specific PCR (MSP), whole genome bisulfite sequencing (BS-Seq), HELP (Hpall tiny fragment Enrichment by Ligation-mediated PCR) assay, ChlP-on-chip assays, restriction landmark genomic scanning. Methylated DNA immunoprecipitation (MeDIP), pyrosequencing of bisulfite treated DNA, molecular break light assay for DNA adenine methyltransferase activity, methyl sensitive Southern Blotting, separation of native DNA into methylated and unmethylated fractions using MethylCpG Binding Proteins (MBPs) and fusion proteins containing just the Methyl Binding Domain (MBD).

[0728] In an embodiment, the modulator cleaves DNA. For example, a modulator can catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. In some embodiments, the modulator (e.g., DNase I) cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3'. In other embodiments, the modulator (e.g., DNase II) hydrolyzes deoxyribonucleotide linkages in DNA, yielding products with 3'-phosphates. In some embodiments, the modulator comprises endodeoxyribonuclease activity. In other embodiments, the modulator comprises exodeoxyribonuclease activity (e.g., having 3' to 5' or 5' to 3' exodeoxyribonuclease activity). In some embodiments, the modulator recognizes a specific DNA sequence (e.g., a restriction enzyme). In other embodiments, the modulator does not cleave DNA in a sequence-specific manner. A modulator can cleave single-stranded DNA (e.g., having nickase activity), double-stranded DNA, or both.

[0729] n an embodiment, modulator affects, e.g., alters or preserves, tertiary or quaternary DNA structure. For example, the modulators described herein can modulate tertiary structure, e.g., handedness (right or left), length of the helix turn, number of base pairs per turn, and/or difference in size between the major and minor grooves. n some embodiments, the modulator mediates the formation of B-DNA, A-DNA, and/or Z-DNA. The modulators described herein can also modulate quaternary structure, e.g., the interaction of DNA with other molecules (DNA or non-DNA molecules, e.g., histones), e.g., in the form of chromatin. In some embodiments, the modulator that mediate or modify tertiary or quatemary DNA structure comprises DNA helicases activity or modulates DNA helicase activity.

[0730] In some embodiments, the modulator promotes or inhibits DNA damage response and/or repair. For example, a modulator can promote one or more DNA damage response and repair mechanisms, e.g., direct reversal, base excision repair (BER), nucleotide excision repair (NER) (e.g., global genomic repair (GG-NER), transcription-coupled repair (TC-NER)), mismatch repair (MMR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, and/or translesion synthesis (TLS). In some embodiments, a modulator promotes the step of damage recognition. In other embodiments, a modulator promotes the step of DNA repair.

[0731] Aberrant DNA damage repair is associated with various diseases and conditions, e.g., aging, hereditary DNA repair disorders, and cancer. For example, DNA repair gene mutations that can increase cancer risk include, e.g., BRCA1 and BRCA2 (e.g., involved in homologous recombination repair (HRR) of double-strand breaks and daughter strand gaps, e.g., in breast and ovarian cancer); ATM (e.g., different mutations reduce HRR, single strand annealing (SSA), NHEJ or homology-directed DSBR (HDR), e.g., in leukemia, lymphoma, and breast cancer), NBS (e.g., involved in NHEJ, e.g., in lymphoid malignancies); MRE1 1 (e.g., involved in HRR, e.g., in breast cancer); BLM (e.g., involved in HRR, e.g., iri leukemia, lymphoma, colon, breast, skin, auditory canal, tongue, esophagus, stomach, tonsil, larynx, lung, and uterus cancer); WRN (e.g., involved in HRR, NHEJ, long-patch BER, e.g., in soft tissue sarcomas, colorectal, skin, thyroid, and pancreatic cancer); RECQ4 (RECQL4) (e.g., involved in HRR, e.g., causing Rothmund-Thomson syndrome (RTS), RAPAD1L1NO syndrome or Bailer Gerold syndrome, cutaneous carcinomas, including basal cell carcinoma, squamous cell carcinoma, and Bowen's disease); FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN (e.g., involved in HRR and TLS, e.g., in leukemia, liver tumors, solid tumors in many locations), XPC and XPE(DDB2) (e.g., involved in NER(GGR type), e.g., in skin cancer (melanoma and non-melanoma)); XPA, XPB, XPD, XPF, and XPG (e.g., involved in NER (both GGR type and TCR type), e.g., in skin cancer (melanoma and non-melanoma) and central nervous system); XPV(POLH) (e.g., involved in TLS, e.g., in skin cancer (melanoma and non-melanoma)); hMSH2, hMSH6, hMLHl, and hPMS2 (involved in MMR, e.g., in colorectal, endometrial and ovarian cancer); MUTYH (e.g., involved in BER of A mispaired with 80H-dG, as well as mispairs with G, FapydG and C, e.g., in colon cancer)

[0732] Modulators can be used to treat a disease or condition associated with aberrant DNA damage repair, e.g., by modulating one or more DNA damage repair mechanisms described herein.

[0733] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in direct reversal, e.g., methyl guanine methyl transferase (MGMT).

[0734] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in BER, e.g., DNA glycosylase, AP endonuclease, DNA polymerase, DNA ligase.

[0735] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in GG-NER, e.g., XPC, HR23b, CAK, TFIIH, XPA, RPA, XPG, XPF, ERCC1, TFIIH, PCNA, RFC, ADN Pol, and Ligase I.

[0736] In some embodiments, the modulator is selected from ,or modulates, one or more proteins involved in TC-NER, e.g., CSB, XPA, RPA, XPG, XPF, ERCC1, CSA-CNS, TFIIH, CAK, PCNA, RFC, Ligase I, and RNA Polymerase II.

[0737] In some embodiments, the modulator is selected from, or modulates, one or more DNA mismatch repair proteins.

[0738] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in NHEJ, e.g., Ku70/80, DNA-PKcs, DNA Ligase IV, XRCC4, XLF, Artemis, DNA polymerase mu, DNA polymerase lambda, PNKP, Aprataxin, and APLF. [0739] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in homologous recombination, e.g., as described herein.

[0740] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in TLS, e.g., DNA polymerase eta, iota, kappa, zeta, and PCNA.

[0741] In an embodiment, a modulator can modulate global response to DNA damage, e.g., D A damage checkpoints and/or transcriptional responses to DNA damage. For example, DNA damage checkpoints can occur at the Gl/S and G2 boundaries. An intra-S checkpoint can also exist. Checkpoint activation can be modulated by two master kinases, ATM and ATR. ATM can respond to DNA double-strand breaks and disruptions in chromatin structure and ATR can respond to stalled replication forks. These kinases can phosphorylate downstream targets in a signal transduction cascade, e.g., leading to cell cycle arrest. A class of checkpoint mediator proteins (e.g., BRCA1, MDC1, and 53BP1), which transmit the checkpoint activation signal to downstream proteins, can be modulated. Exemplary downstream proteins that can be modulated include, e.g., p53, p21, and cyclin/cyclin-dependent kinase complexes.

[0742] In some embodiments, the modulator modulates nuclear DNA damage response and repair. In other embodiments, the modulator modulates mitochondrial DNA damage response and repair.

[0743] In some embodiments, the modulator promotes or inhibits DNA replication. For example, a modulator can promote or inhibit one or more stages of DNA replication, e.g., initiation (e.g., assembly of pre-replicative complex and/or initiation complex), elongation (e.g., formation of replication fork), and termination (e.g., formation of replication fork barrier). In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in initiation, e.g., the origin recognition complex (ORC), CDC6, CDT1, minichromosome maintenance proteins (e.g., MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, and MCM10),

CDC45, CD , DDK, CDC 0 , CDC 02, CDC 103, and CDC 105. In some embodiments, the modulator is selected from, or modulates ,one or more proteins involved in elongation, e.g., DNA hel icases, DNA polymerase, PCNA, CDC45-MCM-GINS helicase complex, and Replication Factor C complex.

[0744] In some embodiments, the modulator is selected, from or modulates, one or more proteins involved in termination, e.g., type II topoisomerase and telomerase. In some embodiments, the modulator is selected from, or modulates, one or more replication checkpoint proteins, e.g., ATM, ATR, ATRIP, TOPBP1, RAD9, HUS1, Radl, and CH 1.

[0745] In some embodiments, the payload comprises a modulator of nuclear DNA replication. In other embodiments, the modulator promotes or inhibits mitochondrial DNA replication.

[0746] Defects in DNA replication can be associated with various diseases and conditions, e.g., cancer and neurological diseases (e.g., Alzheimer's disease). Defects in mitochondrial DNA replication can also be associated with diseases and conditions, e.g., mtDNA depletion syndromes (e.g., Alpers or early infantile hepatocerebral syndromes) and mtDNA deletion disorders (e.g., progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)). A modulator can be used to treat a disease or condition associated with aberrant DNA replication, e.g., by modulating DNA replication as described herein.

[0747] Exemplary endogenous or exogenous modulators of DNA structure are described herein, e.g., in Table VI-3.

Tabic VI-3 DNAH 17-AS 1 DNAH17 antisense RNA 1 DNAI1 dynein, axonemal, intermediate chain 1 DNAI2 dynein, axonemal, intermediate chain 2 DNAJB8-AS1 DNAJB8 antisense RNA 1 DNAJC3-AS1 DNAJC3 antisense RNA 1 (head to head) DNAJC9-AS1 DNAJC9 antisense RNA 1 DNAJC25- DNAJC25-GNG 10 readthrough GNG10 DNAJC27-AS1 DNAJC27 antisense RNA 1 DNAL1 dynein, axonemal, light chain 1 DNAL4 dynein, axonemal, light chain 4 DNALI dynein, axonemal, light intermediate chain 1 DNASE1 deoxyribonuclease 1 DNASE1 L 1 deoxyribonuclease I-like 1 DNASE1L2 deoxyribonuclease I-like 2 DNASE1L3 deoxyribonuclease I-like 3 DNASE2 deoxyribonuclease II, lysosomal DNASE2B deoxyribonuclease II beta CD226 CD226 molecule FAM 120A family with sequence similarity 120A GAK cyclin G associated kinase GCFC2 GC-rich sequence DNA-binding factor 2 MC 0 minichromosome maintenance complex component 0 PR DC protein kinase, DNA-activated, catalytic polypeptide SACS spastic ataxia of Chaiievoix-Saguenay (sacsin) SCNN1D sodium channel, non-voltage-gated 1, delta subunit SPATS2L spermatogenesis associated, serine-rich 2-like MT7SDNA mitochondrially encoded 7S DNA DCLRE1A DNA cross-link repair 1A DCLRE1 B DNA cross-link repair IB DCLRE1C DNA cross-link repair 1C DDIT3 DNA-damage-inducible transcript 3 DDIT4 DNA-damage-inducible transcript 4 DD1T4L DNA-damage-inducible transcript 4-like DFFA DNA fragmentation factor, 45kDa, alpha polypeptide DFFB DNA fragmentation factor, 40kDa, beta polypeptide (caspase-activated DNase) DMAP1 DNA methyltransferase 1 associated protein 1 DMC1 DNA meiotic recombinase 1 DNMT1 DNA (cytosine-5-)-methyltransferase 1 DNMT3A DNA (cytosine-5-)-methyltransferase 3 alpha DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta DNMT3L DNA (cytosine-5-)-methyltransferase 3-like DNTT DNA nucleotidylexotransferase DRAM 1 DNA-damage regulated autophagy modulator 1 DRAM2 DNA-damage regulated autophagy modulator 2 DSCC1 DNA replication and sister chromatid cohesion 1 ZBP1 Z-DNA binding protein 1 SON SON DNA binding protein TARDBP TAR DNA binding protein BMF Bcl2 modifying factor CENPBD1 CENPB DNA-binding domains containing 1 UNG uracil-DNA glycosylase PDRG1 p53 and DNA-damage regulated 1 TDG thymine-DNA glycosylase TDP1 tyrosyl-DNA phosphodiesterase 1 TDP2 tyrosyl-DNA phosphodiesterase 2 AHDC1 AT hook, DNA binding motif, containing 1 GMNN geminin, DNA replication inhibitor PRIM1 primase, DNA, polypeptide 1 (49kDa) PRIM2 primase, DNA, polypeptide 2 (58kDa) HELB helicase (DNA) B LIG 1 ligase I, DNA, ATP-dependent SUMF1 sulfatase modifying factor 1 SUMF2 sulfatase modifying factor 2 LIG4 ligase IV, DNA, ATP-dependent LIG3 ligase III, DNA, ATP-dependent MDC1 mediator of DNA-damage checkpoint 1 MMS22L MMS22-like, DNA repair protein POLAl polymerase (DNA directed), alpha 1, catalytic subunit POLA2 polymerase (DNA directed), alpha 2, accessory subunit POLB polymerase (DNA directed), beta POLD1 polymerase (DNA directed), delta 1, catalytic subunit POLD2 polymerase (DNA directed), delta 2, accessory subunit POLD3 polymerase (DNA-directed), delta 3, accessory subunit POLD4 polymerase (DNA-directed), delta 4, accessory subunit POLDIP2 polymerase (DNA-directed), delta interacting protein 2 - POLDIP3 polymerase (DNA-directed), delta interacting protein 3 POLE polymerase (DNA directed), epsilon, catalytic subunit POLE2 polymerase (DNA directed), epsilon 2, accessory subunit POLE3 polymerase (DNA directed), epsilon 3, accessory subunit POLE4 polymerase (DNA-directed), epsilon 4, accessory subunit POLG polymerase (DNA directed), gamma POLG2 polymerase (DNA directed), gamma 2, accessory subunit POLH polymerase (DNA directed), eta POLI polymerase (DNA directed) iota POLK polymerase (DNA directed) kappa POLL polymerase (DNA directed), lambda POLM polymerase (DNA directed), mu POLN polymerase (DNA directed) nu POLO polymerase (DNA directed), theta ID1 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein ID2 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein ID4 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein OGG1 8-oxoguanine DNA glycosylase MSANTD1 Myb/SANT-like DNA-binding domain containing 1 MSANTD2 Myb/SANT-like DNA-binding domain containing 2 MSANTD3 Myb/SANT-like DNA-binding domain containing 3 MSANTD4 Myb/SANT-like DNA-binding domain containing 4 with coiled-coils P1F1 PIF1 5'-to-3' DNA helicase TONSL tonsoku-like, DNA repair protein MPG N-methylpurine-DNA glycosylase TOPI topoisomerase (DNA) I TOP1MT topoisomerase (DNA) I, mitochondrial TOP2A topoisomerase (DNA) II alpha 170kDa TOP2B topoisomerase (DNA) II beta 180kDa TOP3A topoisomerase (DNA) III alpha TOP3B topoisomerase (DNA) III beta TOPBP1 topoisomerase (DNA) II binding protein 1 DDB 1 damage-specific DNA binding protein 1, 127kDa DDB2 damage-specific DNA binding protein 2, 48kDa SSBP1 single-stranded DNA binding protein 1, mitochondrial SSBP2 single-stranded DNA binding protein 2 SSBP3 single stranded DNA binding protein 3 SSBP4 single stranded DNA binding protein 4 GADD45A growth arrest and DNA-damage-inducible, alpha GADD45B growth arrest and DNA-damage-inducible, beta GADD45G growth arrest and DNA-damage-inducible, gamma GADD45GIP1 growth arrest and DNA-damage-inducible, gamma interacting protein 1 MGMT O-6-methylguanine-DNA methyltransferase REV1 REV1, polymerase (DNA directed) RECOL RecQ protein-like (DNA helicase Ql-like) CCDC6 coiled-coil. domain containing 6 KLRK1 killer cell lectin-like receptor subfamily K, member 1 N6AMT1 N-6 adenine-specific DNA methyltransferase 1 (putative) N6AMT2 N-6 adenine-specific DNA methyltransferase 2 (putative) POLR2A polymerase (RNA) 1 (DNA directed) polypeptide A, 220kDa POLR2B polymerase (RNA) II (DNA directed) polypeptide B, 140kDa POLR2C polymerase (RNA) II (DNA directed) polypeptide C, 33kDa POLR2D polymerase (RNA) II (DNA directed) polypeptide D POLR2E polymerase (RNA) II (DNA directed) polypeptide E, 25kDa POLR2F polymerase (RNA) II (DNA directed) polypeptide F POLR2G polymerase (RNA) II (DNA directed) polypeptide G POLR2H polymerase (RNA) II (DNA directed) polypeptide H POLR2I polymerase (RNA) II (DNA directed) polypeptide I, 14.5kDa POLR2J polymerase (RNA) II (DNA directed) polypeptide J, 13.3kDa POLR2J2 polymerase (RNA) II (DNA directed) polypeptide J2

POLR2J3 polymerase (RNA) 11 (DNA directed) polypeptide J3 POLR2 polymerase (RNA) II (DNA directed) polypeptide , 7.0kDa POLR2L polymerase (RNA) II (DNA directed) polypeptide L, 7.6kDa POLR2M polymerase (RNA) II (DNA directed) polypeptide M TRDMTl tRNA aspartic acid methyltransferase 1 CHD1 chiOmodomain helicase DNA binding protein 1 CHD1 L chromodomain helicase DNA binding protein 1-like CHD2 chromodomain helicase DNA binding protein 2 CHD3 chromodomain helicase DNA binding protein 3 CHD4 chiOmodomain helicase DNA binding protein 4 CHD5 chromodomain helicase DNA binding protein 5 CHD6 ch -omodomain helicase DNA binding protein 6 CHD7 chromodomain helicase DNA binding protein 7 CHD8 chiOmodomain helicase DNA binding protein 8 CHD9 chromodomain helicase DNA binding protein 9 LLN killin, p53-regulated DNA replication inhibitor POLR3A polymerase (RNA) III (DNA directed) polypeptide A, 155kDa POLR3B polymerase (RNA) III (DNA directed) polypeptide B POLR3C polymerase (RNA) III (DNA directed) polypeptide C (62kD) POLR3D polymerase (RNA) III (DNA directed) polypeptide D, 44kDa POLR3E polymerase (RNA) III (DNA directed) polypeptide E (80kD) POLR3F polymerase (RNA) III (DNA directed) polypeptide F, 39 kDa POLR3G polymerase (RNA) III (DNA directed) polypeptide G (32kD) POLR3GL polymerase (RNA) III (DNA directed) polypeptide G (32kD)-like POLR3H polymerase (RNA) III (DNA directed) polypeptide H (22.9kD) POLR3K polymerase (RNA) III (DNA directed) polypeptide , 12.3 kDa WDHD1 WD repeat and HMG-box DNA binding protein 1 PGAP1 post-GPI attachment to proteins 1 PGAP2 post-GPI attachment to proteins 2 PGAP3 post-GPI attachment to proteins 3 REV3L REV3-like, polymerase (DNA directed), zeta, catalytic subunit CDT1 chromatin licensing and DNA replication factor 1 PANDAR promoter of CD 1A antisense DNA damage activated RNA APEX1 APEX nuclease (multifunctional DNA repair enzyme) 1 CHMP1A charged multivesicular body protein 1A CHMP1 B charged multivesicular body protein IB CHMP2A charged multivesicular body protein 2A CHMP2B charged multivesicular body protein 2B CHMP4A charged multivesicular body protein 4A CHMP4B charged multivesicular body protein 4B CHMP4C charged multivesicular body protein 4C CH P5 charged multivesicular body protein 5 CHMP6 charged multivesicular body protein 6 POLRMT polymerase (RNA) mitochondrial (DNA directed) SPIDR scaffolding protein involved in DNA repair MCIDAS multiciliate differentiation and DNA. synthesis associated cell cycle protein PAPD7 PAP associated domain containing 7 RFX8 RFX family member 8, lacking RFX DNA binding domain DE DEK oncogene NUB1 negative regulator of ubiquitin-like proteins 1 PAXBP1 PAX3 and PAX7 binding protein 1 RAMP receptor (G protein-coupled) activity modifying protein 1 RAMP2 receptor (G protein-coupled) activity modifying protein 2 RAMP3 receptor (G protein-coupled) activity modifying protein 3 RC3H2 ring finger and CCCH-type domains 2 ARHGAP35 Rho GTPase activating protein 35 SMUG1 single-strand-selective monofunctional uracil-DNA glycosylase 1 CXXC1 CXXC finger protein 1 FAM50A family with sequence similarity 50, member A FANCG Fanconi anemia, complementation group G GL13 GLI family zinc finger 3 GTF2H5 general transcription factor IIH, polypeptide 5 LAGE3 L antigen family, member 3 MYCNOS MYCN opposite strand/antisense RNA NFRKB nuclear factor related to kappaB binding protein RAD51D RAD51 paralog D RFX2 regulatory factor X, 2 (influences HLA class II expression) RFXANK regulatory factor X-associated ankyrin-containing protein RRP1 ribosomal RNA processing 1 SPRTN SprT-like N-terminal domain XRCC4 X-ray repair complementing defective repair in Chinese hamster cells 4 CDK1 1A cyclin-dependent kinase 11A CDK1 1B cyclin-dependent kinase 1IB LURAP1 L leucine rich adaptor protein 1-like MAD2L2 MAD2 mitotic arrest deficient-like 2 (yeast) PRDM2 PR domain containing 2, with ZNF domain NABP2 nucleic acid binding protein 2 NABP1 nucleic acid binding protein 1 PPP1R15A protein phosphatase 1, regulatory subunit 15A TATDN1 TatD DNase domain containing 1 TATDN2 TatD DNase domain containing 2 TATDN3 TatD DNase domain containing 3 CEBPB CCAAT/enhancer binding protein (C/EBP), beta INIP E T S3 and NABP interacting protein I TS3 integrator complex subunit 3 SDIM1 stress responsive DNAJB4 interacting membrane protein 1 DHX9 DEAH (Asp-Glu-Ala-His) (SEQ ID NO: 39) box helicase 9 SATBl SATB homeobox 1 FEN1 flap structure-specific endonuclease 1 HCST hematopoietic cell signal transducer TYROBP TYRO protein tyrosine kinase binding protein AFA ankyloblepharon filiforme adnatum C9orfl69 chromosome 9 open reading frame 169 TSP02 translocator protein 2 TCIRG T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V O subunit A3 Clorf61 chromosome 1open reading frame 6 1 HLA-DOA major histocompatibility complex, class II, DO alpha SPINK 13 serine peptidase inhibitor, Kazal type 13 (putative)

[0748] In some embodiments, the payload comprises a modulator of an epigenetic state or characteristic of a component of chromatin, e.g., a chromatin associated protein, e.g., a histone. For example, the epigenetic state or characteristic can comprise histone acetylation, deacetylation, methylation (e.g., mono, di, or tri-methylation), demethylation, phosphorylation, dephosphorylation, ubiquitination (e.g., mono or polyubiquitination), deubiquitination, sumoylation, ADP-ribosylation, deimination, or a combination thereof.

[0749] In some embodiments, the modulator is selected from, or modulates, one or more histone modifying enzymes. In an embodiment, the histone modifying enzyme is a histone methyltransferase (HMT). In some embodiments, the histone modifying enzyme is a histone demethyltransferase (HDMT). In some embodiments, the histone modification enzyme is a histone acetyltransferase (HAT). In some embodiments, the histone modifying enzyme is a histone deacetylase (HDAC). In some embodiments, the histone modification enzyme is a kinase. In some embodiments, the histone modifying enzyme is a phosphatase. In some embodiments, the histone modifying enzyme is ubiquitin-activating enzymes (Els), ubiquitin- conjugating enzymes (E2s), or ubiquitin ligases (E3s). In some embodiments, the histone modifying enzyme is a deubiquitinating (DUB) enzyme.

[0750] In some embodiments, histone modifications involved in regulation of gene transcription are modulated. For example, mono-methylation of H3K4, H3K9, H3K27, H3K79, H4K20, H2B 5, di-methylation of H3K79, tri-methylation of H3K4, H3K79, H3K36, and acetylation of H3K9, H3K14, H3K27, can be associated with transcription activation. As another example, di- methylation of H3K9, H3K27, and tri-methylation of H3K9, H3K27, H3K79, H2BK5 can be associated with transcription repression. n some embodiments, the modulator modulates trimethylation of H3 lysine 4 (H3K4Me3) and/or trimethylation of H3 lysine 36 (H3K36Me3), e.g., in active genes. In other embodiments, the modulator modulates trimethylation of H3 lysine 27 (H3K27Me3), di- and tri-methylation of H3 lysine 9 (H3K9Me2/3), and/or trimethylation of H4 lysine 20 (H4K20Me3), e.g., in repressed genes. In some embodiments, the modulator modulates both activating (e.g., H3K4Me3) and repressing (e.g., H3K27Me3) marks, e.g., in stem cells.

[0751] n some embodiments, histone modifications involved in DNA damage response and repair are modulated. For example, the modulators described herein can modulate phosphorylation of H2AX at Serine 139 and/or acetylation of H3 lysine 56 (H3K56Ac).

[0752] Aben ant histone modifications are associated with various diseases and conditions, e.g., cancer, cardiovascular disease, and neurodegenerative disorder. The modulators described herein can be used to treat a disease or condition described herein, e.g., by modulating one or more histone modifications, as described herein.

[0753] Epigenetic changes in histones can be evaluated by art-known methods or as described herein. Exemplary methods for detecting histone modifications include, e.g., chromatin immunoprecipitation (ChIP) using antibodies against modified histones, e.g., followed by quantitative PCR.

[0754] Exemplary endogenous or exogenous modulators of chromatin structure are described herein, e.g., in Table Vl-4.

Table VI-4 KMT1F MT2A lysine (K)-specific methyltransferase TRX1, HRX, ALL- 1, NM_005933 2A HTRX1, CXXC7, MLL1A KMT2B lysine (K)-specific methyltransferase KIAA0304, MLL2, NM_0 14727 2B TRX2, HRX2, WBP7, MLL1B, MLL4 KMT2C lysine (K)-specific methyltransferase KIAA1506, HALR 2C KMT2D lysine (K)-specific methyltransferase ALR, MLL4, 2D CAGL1 14 KMT2E lysine (K)-specific methyltransferase HDCMC04P 2E SETD 1A SET domain containing 1A KIAA0339, Setl, NM_0147 12 KMT2F SETD1B SET domain containing IB KIAA1076, SetlB, XM_037523 MT2G ASH 1L ashl (absent, small, or homeotic)-like huASHl, ASH1, NM_0 18489 (Drosophila) ASH 1LI, KMT2H SETD2 SET domain containing 2 HYPB, HIF-1, NM_014159 KIAA1732, FLJ23184, MT3A NSDl nuclear receptor binding SET domain ARA267, FLJ22263, NM_1 72349 protein 1 KMT3B SMYD2 SET and MYND domain containing HSKM-B, ZMYND14, NM_020197 2 KMT3C SMYD1 SET and MYND domain containing BOP, ZMYND22, XM_097915 1 KMT3D SMYD3 SET and MYND domain containing KMT3E NM_022743 3 DOT1 L DOTl-like histone H3K79 KIAA1814, DOT1, NM_032482 ethy1transferase KMT4 SETD8 SET domain containing (lysine SET8, SET07, PR- NM_020382 methyltransferase) 8 Set7, KMT5A SUV420H suppressor of variegation 4-20 CGI-85, MT5B NM_0 17635 homolog 1 (Drosophila) SUV420H suppressor of variegation 4-20 MGC2705, KMT5C NM_032701 2 homolog 2 (Drosophila) EZH2 enhancer of zeste homolog 2 EZH1, ENX-1, MT6, (Drosophila) KMT6A EZH 1 enhancer of zeste homolog 1 KIAA0388, MT6B NM_001991 (Drosophila) SETD7 SET domain containing (lysine KIAA1717, SET7, NM_030648 methyltransferase) 7 SET7/9, Set9, KMT7 PRDM2 PR domain containing 2, with ZNF RIZ, RIZ1, RIZ2, NM_0 12231 domain MT8, MTB-ZF, HUMHOXY1 HAT1 histone acetyltransferase 1 KAT1 NM_003642 KAT2A K(lysine) acetyltransferase 2A GCN5, PCAF-b NM_021078 KAT2B K(lysine) acetyltransferase 2B P/CAF, GCN5, NM_003884 GCN5L CREBBP CREB binding protein RTS, CBP, KAT3A NM_004380 EP300 El A binding protein p300 p300, AT3B NM_001429 TAFl TAFl RNA polymerase II, TATA NSCL2, TAFII250, NM_004606 box binding protein (TBP)-associated AT4, DYT3/TAF1 factor, 250kDa KAT5 K(lysine) acetyltransferase 5 TIP60, PLIP, cPLA2, NM_006388 HTATIP1, ESA1, ZC2HC5 KAT6A K(lysine) acetyltransferase 6A MOZ, ZC2HC6A NM_006766 KAT6B K(lysine) acetyltransferase 6B querkopf, qkf, Morf, NM_0 12330 MOZ2, ZC2HC6B AT7 K(lysine) acetyltransferase 7 HBOA, HBOl , NM_007067 ZC2HC7 KAT8 K(lysine) acetyltransferase 8 MOF, FLJ 14040, NM_032188 hMOF, ZC2HC8 ELP3 elongator acetyltransferase complex FLJ 10422, KAT9 NM_0 18091 subunit 3 GTF3C4 general transcription factor IIIC, TFIIIC90, KAT12 polypeptide 4, 90kDa NCOA1 nuclear receptor coactivator 1 SRC1, F-SRC-1, NM_147223 NCoA-1, KAT13A, RIP 160, bHLHe74 NCOA3 nuclear receptor coactivator 3 RAC3, AIB1, ACTR, NM_006534 p/CIP, TRAM-1, CAGH16, TNRC16, KAT13B, bHLHe42, SRC-3, SRC3 NCOA2 nuclear receptor coactivator 2 TIF2, GRIP1, NCoA-2, AT 13C, bHLHe75 CLOCK clock circadian regulator KIAA0334, KAT13D, NM_004898 bHLHe8 KDM1A lysine (K)-specific demethylase 1A KIAA0601. BHC1 10, NM_015013 LSD1 KDM1B lysine (K)-specific demethylase B FLJ34109, FLJ33898, NM_ 153042 dJ298J15.2, bA204B7.3, FLJ43328, LSD2 KDM2A lysine (K)-specific demethylase 2A K1AA1004, FBL1 1, NM_0 12308 LILINA, DKFZP434M1735, FBL7, FDO01 15, CXXC8, JHDM1A KDM2B lysine (K)-specific demethylase 2B PCCX2, CXXC2, NM_032590 FbllO, JHDM1B KDM3A lysine (K)-specific demethylase 3A TSGA, KIAA0742, NM_0 18433 JHMD2A KDM3B lysine (K)-specific demethylase 3B KIAA1082, NET22 NM_0 16604 KDM4A lysine (K)-specific demethylase 4A KIAA0677, JHDM3A, NM_0 14663 TDRD14A KDM4B lysine (K)-specific demethylase 4B KIAA0876, TDRD14B NM_015015 KDM4C lysine (K)-specific demethylase 4C GASC1 , KIAA0780, NM_0 15061 TDRD14C KD 4D lysine (K)-specific demethylase 4D FLJ 10251 NM_0 18039 KDM4E lysine (K)-specific demethylase 4E JMJD2E NM_001 161630 KDM5A lysine (K)-specific demethylase 5A NM_005056 KDM5B lysine (K)-specific demethylase 5B RBBP2H1A, PLU-1, NM_006618 CT31 KDM5C lysine (K)-specific demethylase 5C DXS1272E, XE169 NM_004187 KDM5D lysine (K)-specific demethylase 5D ΚΓΑΑ0234 NM_004653 KDM6A lysine (K)-specific demethylase 6A NM_021 140 KDM6B lysine (K)-specific demethylase 6B KIAA0346 XM_043272 JHDM1 D jumonji C domain containing histone KIAA1718 NM_030647 demethylase 1 homolog D (S. cerevisiae) PHF8 PHD finger protein 8 ZNF422, KIAA1 111, NM_015107 JHDM1F PHF2 PHD finger protein 2 KIAA0662, JHDM1E, NM_005392 CENP-35 KDM8 lysine (K)-specific demethylase 8 FLJ 13798 NM_024773 Modulators of Gene Expression

[0755] In an embodiment a payload comprises a modulator of gene expression. A modulator of gene expression can be delivered in vitro, ex vivo, or in vivo.

[0756] In an embodiment, the payload comprises a transcription factor. Transcription factors can bind to specific DNA sequences (e.g., an enhancer or promoter region) adjacent to the genes that they regulate. For example, transcription factors can stabilize or inhibit the binding of RNA polymerase to DNA, catalyze the acetylation or deacetylation of histone proteins (e.g., directly or by recruiting other proteins with such catalytic activity), or recruit coactivator or corepressor proteins to the transcription factor/DNA complex. Modulators of gene expression also include, e.g., any proteins that interact with transcription factors directly or indirectly.

[0757] In an embodiment, the transcription factor is a general transcription factor, e.g., is ubiquitous and interacts with the core promoter region surrounding the transcription start site(s) of many, most or all class IT genes. Exemplary general transcription factors include, e.g., TFI1A, TF1IB, TFIID, TFIIE, TFIIF, and TFIIH. In an embodiment, the transcription factor is an upstream transcription factor, e.g., binds upstream of the initiation site to stimulate or repress transcription. In an embodiment, the transcription factor is a specific transcription factor, e.g., a transcription factor dependent on a recognition sequence present in the proximity of the gene. Exemplary specific transcription factors include, e.g., SP1, AP-1, C EBP, heat shock factor, ATF/CREB, -Myc, OCT-1, and NF-1 .

[0758] In an embodiment, the transcription factor is constitutively active, e.g., a general transcription factor, SP 1, NF- 1, or CCAAT. In other embodiments, the transcription factor is conditionally active, e.g. it requires activation, e.g., developmental (e.g., GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix), signal-dependent (e.g., extracellular ligand (endocrine or paracrine)-dependent, intracellular ligand (autocrine)-dependent (e.g., SREBP, p53, orphan nuclear receptors), cell membrane receptor-dependent (e.g., resident nuclear factors (e.g., CREB, AP-1, Mef2) or latent cytoplasmic factors (e.g., STAT, R-SMAD, NF- Β, Notch, TUBBY, NFAT). [0759] Other exemplary transcription factors are described herein, e.g., in Tables VI-5 and VI-6. (Table VI-5 Transcription Factors, is provided in Annex VI-5)

Table VI-6: Selected Transcription Factors with Anotations angiogenesis, enhanced survival of tumors and immunosuppression.

Thyroid hormone Involved in widely diverse physiological functions, including control receptor(25) of embryonic development, cell differentiation and homeostasis

zf-C2HC(6) Highly transcribed in the developing nervous system. Exemplary diseases include Duane Radial Ray Syndrome. Androgen Exemplary functions include diverse physiological functions, receptorf 1) including control of embryonic development, cell differentiation and homeostasis. Exemplary diseases include X-linked spinal, bulbar muscular atrophy and prostate cancer. CG-1 2 Exemplary functions include calcium signaling by direct binding of calmodulin. CTF/NFK4) Exemplary functions include both viral DNA replication and regulation of gene expression. Exemplary diseases include leukemia, juvenile myelomonbcytic. Fork headf49) Involvement in early developmental decisions of cell fates during embryogenesis. Exemplary diseases include lymphedema-distichiasis, developmental verbal dyspraxia, autoimmune diseases. Homeobox(205) Exemplary functions include involvement in a wide range of critical activities during development. Exemplary diseases include limb malformations, eye disorders, and abnormal head, face, and tooth development. Additionally, increased or decreased activity of certain homeobox genes has been associated with several forms of cancer.

MYB(25 Exemplary functions include regulator of proliferation, differentiation and cell fate. Exemplary diseases include cancer (e.g., oncogenic disease). Oestrogen Control of embryonic development, cell differentiation and receptor( 1) homeostasis. Exemplary diseases include estrogen resistance, familial breast cancer, migrane, myocardial infaction. POU 2 ) Wide variety of functions, related to the function of the neuroendocrine system and the development of an organism. Exemplary diseases include non-syndromic deafness. RHD O) Exemplary diseases include autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS.

T-box(17) TSC22(4) zf-GATA(14) AP-2(5) COE(4) CUT(7) GCM(2) HSF 8) NDT80/P oG l

Other nuclear - receptor(2) P A receptor(3) ROR receptor(4) TEA (4) Tub(5) zf-LITAF-like(2) A ) COUP(3) DMf7) GCR( l) Τ 2 NF- ΥΑ ) OthersO) Progesterone receptor( 1) Runt ) TF bZIP(46) Z T B(48 zf-MIZ(7) bHLH l 06 CP2 7 E 2F 1n GTF2 5 IRF(9) NF-YB/CC2) P53G Prox l(2) SAND(8) TF Otx(3) zf-BED(5) zf-NF-X l 2) C/EBP( 10 CSD 8) Ecdystd receptor(2) HMG 0 MBDf9) N l) PAX(9) Retinoic acid receptor(7) SRF(6) ΤΗΑΡ 2) zf-C2H2(634)

CRX Exemplary diseases include dominant cone-rod dystrophy. Repair mutation. FOCX2 Exemplary diseases include lymphedema-distichiasis. Repair mutation. FO P2 Exemplaiy diseases include developmental verbal dyspraxia. Repair mutation. FOXP3 Exemplary diseases include autoimmune diseases. Repair mutation.

GAT4 Exemplary diseases include congenital heart defects. Repair mutation.

HNF1 through Exemplary diseases include mature onset diabetes of the young HNF6 (MODY), hepatic adenomas and renal cysts. Repair mutation.

LHX3 Exemplaiy diseases include Pituitary disease. Repair mutation.

MECP2 Exemplary diseases include Rett syndrome. Repair mutation.

MEF2A Exemplary diseases include Coronary arteiy disease. Repair mutation.

NARA2 Exemplary diseases include Parkinson disease. Repair mutation.

NF-KB Exemplary diseases include autoimmune arthritis, asthma, septic Activation shock, lung Fibrosis, glomerulonephritis, atherosclerosis, and AIDS. Repair mutation. NF-KB Inhibition Exemplaiy diseases include apoptosis, inappropriate immune cell development, and delayed cell growth. Repair mutation. NIKX2-5 Exemplary diseases include cardiac malformations and atrioventricular conduction abnormalities. NOTCH 1 Exemplary diseases include aortic valve abnormalities.

Modulators of alternative splicing

[0760] In an embodiment, the modulator of gene expression modulates splicing. For example, a modulator can modulate exon skipping or cassette exon, mutually exclusive exons, alternative donor site, alternative acceptor site, intron retention, or a combination thereof. In some embodiments, the modulator is selected from or modulates one or more general or alternative splicing factors, e.g., ASF1. In some embodiments, the modulator modulates alternative splicing (e.g., influences splice site selection) in a concentration-dependent manner.

Modulators of post-transcriptional modification

[0761] In an embodiment, the modulator of gene expression modulates post-transcriptional modification. For example, the modulators described herein can promote or inhibit 5' capping, 3' polyadenylation, and RNA splicing. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in 5' capping, e.g., phosphatase and guanosyl transferase. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in 3' polyadenylation, e.g., polyadenylate polymerase, cleavage and polyadenylation specificity factor (CPSF), and poly(A) binding proteins. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in RNA splicing, e.g., general or alternative splicing factors.

[0762] Exemplary endogenous or exogenous modulators of post-transcriptional modification are described herein, e.g., in Table VI-7.

Table VI-7 PAN: PolyA nuclease CPSF: cleavage/polyadenylation specificity factor CstF: cleavage stimulation factor PAP: polyadenylate polymerase PABP: polyadenylate binding protein PAB2: polyadenylate binding protein 2 CFI: cleavage factor I CFII: cleavage factor Capping/Mcthylation of 5'end RNA triposphatase RNA gluanyltransferase RNA mehyltransferase SAM synthase ubiquitin-conjugating enzyme E2R1 Splicing SR proteins SFRS1 - SFR1 1 which, when bound to exons, tend to promote hnRNP proteins: coded by the following genes:HNRNPA0, HNRNPA1, HNRNPA1L1, HNRNPA1L2, HNRNPA3, HNRNPA2B1, HNRNPAB, HNRNPB1 , HNRNPC, HNRNPCL1 , HNRNPD, HNRPDL, HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, HNRNPK, HNRNPL, HNRPLL, HNRNPM, HNRNPR, HNRNPU, HNRNPUL1, HNRNPUL2, HNRNPUL3 Editing protein ADAR Nuclear export proteins Mex67 Mtr2 Nab2 DEAD-box helicase ("DEAD" disclosed as SEQ ID

. NO: 40) TRANSLATION Initiation eIF4A, eIF4B, elF4E, and eIF4G: Eukaryotic initiation factors GEF: Guanine exchange factor GCN2, PKR, HRI and PERK: Kinases involved in phosphorylating some of the initiation factors Elongation eEFl and eEF2: elongation factors GCN: kinase Termination eRF3: translation termination factor POS -TRANSLATIONAL CONTROL mRNA Degradation ARE-specific binding proteins EXRN 1: exonuclease DCP1, DCP2: Decapping enzymes RCK/p54, CPEB, eIF4E: Translation repression microRNAs and siRNAs: Probably regulate 30% of all genes DICER Ago proteins Nonsense-mediated mRNA decay proteins UPF3A UPF3B eIF4A3 MLN51 Y14/MAGOH MG-1 SMG-5 SMG-6 SMG-7 mRNA Modification Enzymes carry the following functions Phosphorylation N-linked glycosylation Acetylation Amidation Hydroxylation Methylation O-linked glycosylation Ubiquitylation

Inhibitors

[0763] In an embodiment a payload comprises an inhibitor of a payload described above, e.g., an inhibitor of an enzyme transcription factor. In an embodiment a payload comprises an inhibitor of any of the aforementioned payload molecules, processes, activities or mechanisms. In an embodiment, the inhibitor is an antibody molecule (e.g., a full antibody or antigen binding fragment thereof) specific for one of the payload molecules described herein. In an embodiment the inhibitor is a small molecule compound. In some embodiments, the inhibitor is a nucleic acid (e.g., siRNA, shRNA, ribozyme, antisense-oligonucleotide, and aptamer). For example, the payload is an inhibitor of a target, e.g., a trasnscription factor, a post-translational modification enzyme, a post-transcriptional modification enzyme, etc., or a nucleic acid sequence encoding any of the foregoing.

Oithologs

[0764] If a non-human gene or protein is recited herein it is understood that the invention also comprises the human counterpart or ortholog and uses thereof.

VI1A. TARGETS: CELLS

[0765] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., an animal cell or a plant cell), e.g., to deliver a payload, or edit a target nucleic acid, in a wide variety of cells. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Delivery or editing can be performed in vitro, ex vivo, or in vivo.

[0766] In some embodiments, a cell is manipulated by editing (e.g., introducing a mutation or correcting) one or more target genes, e.g., as described herein. In other embodiments, a cell is manipulated by delivering a payload comprising one or more modulators (e.g., as described herein) to the cell, e.g., to a target sequence in the genome of the cell. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vivo. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., ex vivo. '

[0767] In some embodiments, the cells are manipulated (e.g., converted or differentiated) from one cell type to another. In some embodiments, a pancreatic cell is manipulated into a beta islet cell. In some embodiments, a fibroblast is manipulated into an iPS cell. In some embodiments, a preadipocyte is manipulated into a brown fat cell. Other exemplary cells include, e.g., muscle cells, neural cells, leukocytes, and lymphocytes. [0768] In some embodiments, the cell is a diseased or mutant-bearing cell. Such cells can be manipulated to treat the disease, e.g., to correct a mutation, or to alter the phenotyope of the cell, e.g., to inhibit the growth of a cancer cell. For examples, a cell is associated with one or more diseases or conditions describe herein. In some embodiments, the cell is a cancer stem cell. For example, cancer stem cells can be manipulated by modulating the expression of one or more genes selected from: TWIST (TF), ΗΓΡ- a , HER2/neu, Snail (TF), or Wnt.

[0769] In some embodiments, the manipulated cell is a normal cell.

[0770] In some embodiments, the manipulated cell is a stem cell or progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells).

[0771] In some embodiments, the manipulated cells are suitable for producing a recombinant biological product. For example, the cells can be CHO cells or fibroblasts. In an embodiment, a manipulated cell is a cell that has been engineered to express a protein.

[0772] In some embodiments, the cell being manipulated is selected from fibroblasts, monocytic precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts, or preadipocytes. In some embodiments, the cell is manipulated (e.g., converted or differentiated) into muscle cells, erythroid-megakaryocytic cells, eosinophils, iPS cells, macrophages, T cells, islet beta-cells, neurons, cardiomyocytes, blood cells, endocrine progenitors, exocrine progenitors, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, or brown adipocytes.

[0773] In some embodiments, the cell is a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte.

[0774] The Cas9 and gRNA molecules described herein can be delivered to a target cell. In an embodiment, the target cell is a normal cell.

[0775] In an embodiment, the target cell is a stem cell or progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells).

[0776] In an embodiment, the target cell is a CHO cell. [0777] In an embodiment, the target cell is a fibroblast, monocytic precursor, B cells exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, or preadipocyte.

[0778] In an embodiment, the target cell is a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neurons (e.g., a neuron in the brain, e.g., a neuron in the striatum (e.g., a medium spiny neuron), cerebral cortex, precentral gyrus, hippocampus (e.g., a neuron in the dentate gyrus or the CA3 region of the hippocampus), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, putamen, hypothalamus, tectum, tegmentum or substantia nigra), cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte.

[0779] n an embodiment, the target cell is manipulated ex vivo by editing (e.g., introducing a mutation or correcting) one or more target genes and/or modulating the expression of one or more target genes, and administered to the subject.

[0780] Exemplary cells that can be manipulated and exemplary genes that can be modulated are described in Table V -8.

TableVII-8 in vivo. Multiplex. B cells Macrophages Deliver Cas9-activators to C EBPo target activation of transcription factors required for differentiation in vivo. B cells T cells, Delivery Cas9-repressors Pax5 macrophages OR deliver Cas9 endonuclease to ablate Pax5 Exocrine Islet β-cells Deliver Cas9-activators to Pdxl cells target activation of Ngn3 transcription factors MafA required for differentiation in vivo. Multiplex. Fibroblasts Neurons Deliver Cas9-activators to Asell target activation of Bm2 transcription factors Mytll required for differentiation in vivo. Multiplex. fibroblasts cardiomyocytes Deliver Cas9-activators to Gata4 target activation of Mef2c transcription factors Tbx5 required for differentiation in vivo. Multiplex. Fibroblasts Blood cells Deliver Cas9-activators to Oct4 target activation of transcription factors required for differentiation in vivo. Fibroblasts cardiomyocytes Deliver Cas9-activators to Oct4 target activation of Sox2 transcription factors lf4 required for differentiation in vivo. Multiplex. Pancreatic Endocrine Deliver Cas9-activators to Ngn3 progenitor progenitor target activation of transcription factors required for differentiation in vivo. Pancreatic .Exocrine Deliver Cas9-activators to P48 progenitor progenitor target activation of transcription factors required for differentiation in vivo. Pancreatic Duct Deliver Cas9-activators to Hnf6/OC-l progenitor target activation of transcription factors required for differentiation in vivo. Pancreatic acinar Deliver Cas9-activators to Ptfla progenitor target activation of Rpbjl transcription factors required for differentiation in vivo. Multiplex. Endocrine cell Deliver Cas9-activators to Foxa2 progenitor target activation of Nkx2.2 (to make transcription factors Pax6 glucagon) required for differentiation Arx in vivo. Multiplex. Endocrine β cell Deliver Cas9-activators to Mafa progenitor target activation of Pd (to make transcription factors Hlxb9 insulin) required for differentiation Pax4 in vivo. Multiplex. Pax6 Isll Nkx2.2 Nkx6.1 Endocrine δ cell Deliver Cas9-activators to Pax4 progenitor target activation of Pax6 (to make transcription factors somatostatin) required for differentiation in vivo. Multiplex. Endocrine PP cell Deliver Cas9-activators to Nkx2.2 progenitor target activation of (to make transcription factors pancreatic required for differentiation polypeptide) in vivo. Hepatoblast hepatocyte Deliver Cas9-activators to Hnf4 target activation of transcription factors required for differentiation in vivo. Hepatoblast Cholangiocyte Deliver Cas9-activators to Hnf6/OC-l target activation of transcription factors required for differentiation in vivo. Myoblasts Brown adipocyte Deliver Cas9-activators to PRDM16 target activation of C/EBP transcription factors PGClCC required for differentiation PPARy in vivo. Multiplex. preadipocytes Brown adipocyte Deliver Cas9-activators to PRDM16 target activation of C EBP transcription factors required for differentiation in vivo. Multiplex.

Table VII-9: Exemplary cells for manipulation Pancreatic cells, e.g., beta cells Muscle cells Adipocytes Pre-adipocytes Neural cells Blood cells Leukocytes Lymphocyes B cells T cells

Table V - 0: Exemplaiy stem cells for manipulation embryonic stem cells non-embryonic stem cells hematopoietic stem cells adipose stem cells germline stem cells lung stem cells neural stem cells Table VII-1 1: Exemplaiy cancer cells for manipulation lung cancer cells breast cancer cells skin cancer cells brain cancer cells, pancreatic cancer cells hematopoietic cancer cells liver cancer cells kidney cancer cells ovarian cancer cells

Table VTI-12: Exemplaiy non-human cells for manipulation Table VII- 12 Non-human cells for manipulation Plant cells, e.g., crop cells, e.g., corn, wheat, soybean, citrus or vegetable cells

Animal cells, e.g., a cow, pig, horse, goat, dog or cat cell

[0781] Exemplary endogenous or exogenous modulators of cancer stem cells (CSCs) are described herein, e.g., in Table VII-13:

Table VII-13

• TWIST 1 (TF) • HIF-la (TF) • HER2/neu • Snail (TF) • Wnt • TGFP • FGF • EGF • HGF • STAT3 (TF) • Notch • P63 (TF) • PI3K)/AKT • Hedgehog • NF- Β (TF) • ATF2 (TF) • miR-200 and miR-34 • P53 (TF) • E-cadherin • Transcription factors that inhibit E-cadherin directly . • ZEB1 • ZEB2 • E47 • KLF8 • Transcription factors that inhibit E-cadherin directly • TCF4 • SIX1 • FOXC2 • G-CSF and CD34 in AML • PML and FOXO in CML • CD133 in glioblastoma multiforme, osteosarcoma, Ewing's sarcoma, endometrial, hepatocellular, colon and lung carcinomas and ovarian and pancreatic adenocarcinoma • CD44 in head and neck cancer, prostate, gastric and colorectal carcinoma stem cells • CD34 in leukemia • CD38 in leukemia β IL3Ra in leukemia • EpCAM in colon carcinoma and pancreatic adenocarcinoma stem cells • ALDH in melanoma, colorectal, breast, prostate and squamous cell carcinomas, pancreatic adenocarcinoma, and osteosarcoma ■ MAP2 in melanoma t a6-integrin in glioblastoma • SSEA-1 in gliobalstoma • CD24 in breast cancer and other tumors

[0782] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to increase cell engraftment, e.g., to achieve stable engraftment of cells into a native microenvironment. The engrafting cells, the cells in the native microenvironment, or both, can be manipulated. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid.

[0783] For example, increased efficiency of engraftment of cells can be achieved by: increasing the expression of one or more of the genes described herein, e.g., homing genes, adhesion genes, survival genes, proliferative genes, immune evasion genes, and/or cell protection genes, and/or decreasing the expression of one or more of the genes described herein, e.g., quiescence genes, death/apoptosis genes, and/or immune recognition genes.

[0784] In an embodiment, the gene encodes a homing receptor or an adhesion molecule, e.g., that is involved in directing cell migration towards a tissue in association with a tissue-expressed ligand or region rich in soluble cytokine. In an embodiment, the homing receptor or adhesion molecule is expressed on leukocytes, e.g., lymphocytes or hematopoietic stem cells. In an embodiment, the tissue is bone marrow, e.g., extracellular matrix or stromal cells. In an embodiment, the homing receptor or adhesion molecule is C-X-C chemokine receptor type 4 (CXCR4, also known as fusin or CD184). For example, the expression of CXCR4 on hematopoietic stem cells is upregulated. In an embodiment, the ligand is stromal-derived-factor- 1 (SDF- 1, also known as CXCL1 2). In an embodiment, the homing receptor or adhesion molecule is CD34. In an embodiment, the ligand is addressin (also known as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1)).

[0785] In an embodiment, the gene encodes a receptor, e.g., expressed on a stem cell or progenitor cell, that binds to a ligand, e.g., a chemokine or cytokine. For example, the receptor can be associated with sternness of the cell and/or attracting the cell to a desired microenvironment. In an embodiment, the receptor is expressed on a hematopoietic stem cell. In an embodiment, the receptor is expressed on a neural stem cell. In an embodiment, the receptor is mast/stem cell growth factor receptor (SCFR, also known as proto-oncogene c-Kit or tyrosine-protein kinase Kit or CD1 17). In an embodiment, the ligand is stem cell factor (SCF, also known as steel factor or c-kit ligand). In an embodiment, the receptor is myeloproliferative leukemia virus oncogene (MPL, also known as CD1 10). In an embodiment, the ligand is thrombopoietin (TPO). [0786] n an embodiment, the gene encodes a marker, e.g., that promotes survival or proliferation of the cells expressing that marker, or allows the cells expressing that marker to evade an immune response or to be protected from an adverse environment, e.g., that leads to cell death. For example, cells expressing CD47 (also known as integrin associated protein (IAP) can avoid phagocytosis, e.g., during cell migration. As another example, cells that express BCL2 can be protected from apoptosis. In an embodiment, the cell is a blood cell, e.g., an erythrocyte or leukocyte. In an embodiment, the cell is a hematopoietic stem cell or progenitor cell.

[0787] In an embodiment, the expression of one or more of CXCR4, SDF1, CD1 17, MPL, CD47, or BCL2, in a stem cell or progenitor cell, e.g., a hematopoietic stem cell or progenitor cell, is upregulated.

[0788] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to manipulate (e.g., dictate) the fate of a targeted cell, e.g., to better target specific cell type of interest and/or as a suicide mechanism. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and/or an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Exemplaiy genes that can be modulated include, e.g., one or more of chemotherapy resistance genes, chemotherapy sensitivity genes, antibiotic resistance genes, antibiotic sensitivity genes, and cell surface receptor genes, e.g., as described herein.

[0789] In an embodiment, a chemotherapy resistance gene, a chemotherapy sensitivity gene, an antibiotic resistance gene, and/or an antibiotic sensitivity gene is modulated, e.g., such that modified or undesirable cells (e.g., modified or undesirable hematopoietic stem cells (HSCs), e.g., in bone marrow) can be reduced or removed, e.g., by chemotherapeutic or antibiotic treatment.

[0790] For example, genes or gene products that modulate (e.g., increase) chemotherapy resistance or antibiotic resistance can be delivered into the cells. Cells modified by the chemotherapy or antibiotic resistance gene or gene product can have a higher (e.g., at least about 2, 3, 4, 5, 6, 7, 8 9, 10, 25, 50, 75, or 100 fold higher) survival rate than cells without such modification after chemotherapeutic or antibiotic treatment. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vivo. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vilro or ex vivo. In an embodiment, the chemotherapy resistance gene is a gene encoding 0 -alkylguanine DNA alkyltransferase (MGMT). In an embodiment, the chemotherapy comprises temozolomide.

[0791] As another example, genes or gene products that modulate (e.g., increase) chemotherapy sensitivity or antibiotic sensitivity can be delivered into the cells. The genes or gene products that confer chemotherapy sensitivity or antibiotic sensitivity can be used as suicide signals, e.g., causing apoptosis of the cells. Cells modified by the chemotherapy or antibiotic sensitivity gene or gene product can have a lower (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 fold lower) survival rate than cells without such modification after chemotherapeutic or antibiotic treatment. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vivo. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vilro or ex vivo.

[0792] The method described herein can be used to select or enrich cells that have a modified or desired phenotype, e.g., chemotherapy resistance and/or antibiotic resistance. The method described herein can also be used to remove or reduce the number of cells that have a modified or undesired phenotype, e.g., chemotherapy sensitivity and/or antibiotic sensitivity. For example, cells that exhibit an undesired effect, e.g., an off-target effect or a cancer phenotype, e.g., caused by editing of a nucleic acid in an undesired genomic location or cell type, can be removed.

[0793] n an embodiment, a cell surface receptor gene is modulated (e.g., the expression of the cell surface receptor is increased or decreased), such that a therapeutic agent (e.g., a therapeutic antibody) can be used to target a cell (e.g., to kill the cell) that has increased or decreased expression of the cell surface receptor. In an embodiment, the cell surface receptor is CD20. In some embodiments, the therapeutic antibody is Rituximab.

[0794] In an embodiment, the cell surface receptor is selected from, e.g., CD52, VEGFR, CD30, EGFR, CD33, or ErbB2. In an embodiment, the therapeutic antibody is selected from, e.g., Alemtuzumab, Rituximab, Cetuximab, Panitumumab, Gentuzaumab, and Trasruzumab. In an embodiment, the cell surface receptor is CD52 and the therapeutic antibody is Alemtuzumab. In an embodiment, the gene encodes VEGF and the therapeutic antibody is Rituximab. In an embodiment, the cell surface receptor is EGFR and the therapeutic antibody is Cetuximab or Panitumumab. In an embodiment, the cell surface receptor is CD33 and the therapeutic antibody is Gentuzaumab. In an embodiment, the cell surface receptor is ErbB2 and the therapeutic antibody is Trastuzumab.

[0795] In an embodiment, the expression or activity of the Cas9 molecule and/or the gRNA molecule is induced or repressed, e.g., when the cell is treated with a drug, e.g., an antibiotic, e.g., in vivo. For example, the induction or repression of the expression or activity of the Cas9 molecule and/or the gRNA molecule can be used to reduce toxicity and/or off-target effects, e.g., in certain tissues. In an embodiment, the expression of the Cas9 molecule, the gRNA molecule, or both, is driven by an inducible promoter. In an embodiment, binding of a drug (e.g., an antibiotic) to the Cas9 molecule and/or the gRNA molecule activates or inhibits the activity of the Cas9 molecule and/or the gRNA molecule. In an embodiment, the drug (e.g., antibiotic) is administered locally. In an embodiment, the cell treated with the drug (e.g., antibiotic) is located

in the eye, ear, nose, mouth, or skin.

[0796] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., in directed enzyme prodrug therapy (DEPT). Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid.

[0797] Directed enzyme prodrug therapy (DEPT) uses enzymes artificially introduced into the body to convert prodrugs, which have no or poor biological activity, to the active form in the desired location within the body. For example, directed enzyme prodrug therapy can be used to reduce the systemic toxicity of a drug, by achieving high levels of the active drug only at the desired site.

[0798] In an embodiment, an enzyme required for prodrug conversion or a gene encoding such an enzyme is delivered to a target cell, e.g., a cancer cell. For example, the enzymes or genes can be delivered by a method described herein. In an embodiment, the gene encoding the enzyme required for prodrug conversion is delivered by a viral vector.

[0799] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to improve immunotherapy, e.g. cancer immunotherapy. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Exemplary genes that can be modulated include, e.g., one or more genes described herein, e.g., PD-L1 and/or PD-L2 genes.

V . TARGETS: PATHWAYSAN GENES

[0800] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate one, two, three or more, elements or a pathway, e.g., by targeting sequences that encode an RNA or protein of a pathway, or sequences that control the expression of an RNA or protein of a pathway. In an embodiment, an element of a first pathway and an element of a second pathway are manipulated. In an embodiment, manipulation comprises delivery of a payload to, or editing, a target nucleic acid. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Delivery or editing can be performed in vitro, ex vivo, or in vivo.

[0801] An element of a pathway can be up or down regulated, e.g., the expression of a gene encoding a protein of a pathway can be increased or decreased. The increase or decrease can be effected by delivery of a payload (e.g., a transcription factor or inhibitor of a transcription factor) or by editing a target nucleic acid (e.g., the use of a template nucleic acid to alter a sequence, e.g., correct or introduce a mutation, in e.g., a control or coding region).

[0802] Exemplary pathways comprise pathways associated with: cell proliferation; cell cycle; carbon metabolism; energy metabolism; glycolysis, anerobic respiration, anerobic respiration; transmembrane signal transduction, angiogenesis, DNA.replication or repair, or pain.

[0803] Exemplary pathways and genes are discussed herein. It will be understood that a pathway or gene can be associated with one or more aspect of cell or organismal function, e.g., a pathway or gene can be involved in both cancer and energy metabolism. Manipulation of a pathway or gene is not limited to the exemplary cell or organismal function listed below. In an embodiment a pathway is associated with one or more diseases or conditions. [0804] In an embodiment, the pathway is associated with cancer, e.g., associated with proliferation (e.g., RAF pathway), evading growth repressors, resisting cell death, enabling replicative immortality/aging, inducing angiogenesis, activating invasion and metastasis, energy metabolism and evading, cancer stem cells, cytokine-receptor interactions, or tumor suppressors. In some embodiments, the pathway is associated with cell cycle control. In some embodiments, the pathway is associated with angiogenesis.

[0805] Pathways and genes associated with cancer are described herein, e.g., include the following:

Table VII-14 Target Genes from Selected Pathways FAK Proliferation Down PKA Proliferation Down RAC Proliferation Down ALK Proliferation Mutated in a subset (2-7%) of lung cancers Rb Evading growth Up suppressors/ pro- apoptotic P53 Evading growth Mutation in colon, lung, esophagus, Up suppressors/ pro- breast, liver, brain reticuloendothelial apoptotic tissues, and hemopoietic tissues APC Evading growth Mutations found in colon and intestine suppressors/ pro- apoptotic CD 4/6 Evading growth Up suppressors/ pro- apoptotic I 4B Evading growth Up suppressors/ pro- apoptotic CD 2 Evading growth Up suppressors/ pro- apoptotic WNT Evading growth Up suppressors/ pro- apoptotic WAF1 Evading growth Up suppressors/ pro- apoptotic Frizzled Evading growth Up suppressors/ pro- apoptotic VHL Evading growth Mutated in all clear cell renal Up suppressors/ pro- carcinomas apoptotic Fas ligand Resisting cell death/ Down anti-apoptotic Fas receptor Resisting cell death/ Down anti-apoptotic Caspase 8 Resisting cell death/ Down anti-apoptotic Caspase 9 Resisting cell death/ Down anti-apoptotic Bcl-2 Resisting cell death/ Correct mutation large deletion in Down anti-apoptotic follicular lymphoma, breast prostate CLL, melanoma Bcl-xL Resisting cell death/ Down anti-apoptotic Bcl-w Resisting cell death/ Down anti-apoptotic Mcl-1 Resisting cell death/ Down anti-apoptotic Bax Resisting cell death/ Down anti-apoptotic Bak Resisting cell death/ Down anti-apoptotic IGF-1 Resisting cell death/ Down anti-apoptotic Puma Resisting cell death/ Down anti-apoptotic Bi Resisting cell death/ Down anti-apoptotic Beclin-1 Resisting cell death/ Down anti-apoptotic TGF-b Enabling replicative immortality/aging Telomerase/TERT Enabling replicative Down immortality/aging ATAD2 Enabling replicative immortality/aging DAF-2 Enabling replicative immortality/aging SRT Enabling replicative immortality/aging Eph-A/B Inducing angiogenesis Down Robo Inducing angiogenesis Down Neuropilin Inducing angiogenesis Down Notch Inducing angiogenesis Down Endostatin Inducing angiogenesis Down Angiostatin Inducing angiogenesis Down FGF family Inducing angiogenesis Down Extracellular Inducing angiogenesis Down matrix-degrading proteases (e.g., MMP-2 & MMP- 9) VEGF-A Inducing angiogenesis Down TSP- 1 Inducing angiogenesis Down VEGFR-1 Inducing angiogenesis Down VEGFR-2 Inducing angiogenesis Down VEGFR-3 Inducing angiogenesis Down NF2 Activating invasion and Down metastasis L Activating invasion and Up- regulated in multiple cancer, Down metastasis including intestine Snail Activating invasion and Down metastasis Slug Activating invasion and Down metastasis Twist Activating invasion and Down metastasis Zebl/2 Activating invasion and Down metastasis CCLR5 Activating invasion and Down metastasis cysteine cathepsin Activating invasion and Down protease family metastasis Extracellular Activating invasion and Down matrix-degrading metastasis proteases (e.g., MMP-2 & MMP- 9) EGF Activating invasion and Down metastasis CSF-1 Activating invasion and metastasis PP2 Energy metabolism Down elF4E Energy metabolism Down RS Energy metabolism Down PI 3CA Energy metabolism Mutated in many breast, bladder Down cancers and hepatocellular carcinoma BAP1 Energy metabolism Mutated in renal cell carcinoma Down TWIST (TF) Cancer Stem Cells Down HIF- l c Cancer Stem Cells Over expressed in renal cell carcinoma Down HER2/neu Cancer Stem Cells Down Snail (TF) Cancer Stem Cells Down Wnt Cancer Stem Cells Down EPCAM Cancer Stem Cells Overexpressed in breast, colon, uterus Down and other cancers EGF Cytokine-receptor Down interactions TGFa Cytokine-receptor Down interactions PDGF Cytokine-receptor Down IGF-1 interactions ILTLG FLT3LG Cytokine-receptor Down interactions HGF Cytokine-receptor Down interactions FGF Cytokine-receptor Down interactions EGFR Cytokine-receptor Mutated in lung cancer (40% of all Down interactions Asians and 10-15% of all Caucasians) ERBB2 Cytokine-receptor Down interactions PDGFR Cytokine-receptor Down interactions IGFR Cytokine-receptor Down interactions c-KlT Cytokine-receptor Down interactions FLT3 Cytokine-receptor Down interactions MET Cytokine-receptor Down interactions FGFR Cytokine-receptor Mutations in bladder cancer Down interactions D A damage and genomic instability DNMT1 Methyl transferases DNMT2 Methyl transferases DNMT3a Methyl transferases DNMT3b Methyl transferases H3K9Me3 Histone methylation H3K27Me Histone methylation Lsh Helicase activity BLM Helicase activity Bloom's syndrome >Cancer Correct WRN Helicase activity Werner's syndrome > Cancer Correct RTS Helicase activity Rothmund-Thompson > Cancer Correct XPA through XPG Nucleotide excision Xeroderma pigmentosa repair XPB Nucleotide excision Cockayne's syndrome repair XAB2 Nucleotide excision repair XPD Nucleotide excision Cockayne's syndrome repair TF1IH Nucleotide excision repair RFC Nucleotide excision repair PC A Nucleotide excision repair LIG Nucleotide excision repair Flap Nucleotide excision endonueclease 1 repair MNAT Nucleotide excision repair MMS19 Nucleotide excision repair RAD23A Nucleotide excision repair RAD23B Nucleotide excision repair RPA1 Nucleotide excision repair RPA2 Nucleotide excision repair CCNH Nucleotide excision repair CD 7 Nucleotide excision repair CETN2 Nucleotide excision repair DDB1 Nucleotide excision repair DDB2 Nucleotide excision repair ERCC1 Nucleotide excision repair ATM Recombinational repair NBN Recombinational repair BRCA 1 Recombinational repair Breast, ovarian and pancreatic cancer Correct susceptibility or Up BRCA2 Recombinational repair Breast cancer and ovarian Correct susceptibility or UP RAD51 Recombinational repair RAD52 Recombinational repair WRN Recombinational repair BLM Recombinational repair FANCB Recombinational repair MLH1 Mismatch repair Multiple (including colon and uterus) MLH2 Mismatch repair Multiple (including colon and uterus) MSH2 Mismatch repair MSH3 Mismatch repair MSH4 Mismatch repair MSH5 Mismatch repair MSH6 Mismatch repair Multiple (including colon and uterus) PMS1 Mismatch repair PMS2 Mismatch repair Multiple (including colon and uterus) PMS2L3 Mismatch repair Aging DAF-2 lGF-1 SRT1 Table VII-15

Genes Mutated in Common Cancers

Bladder FGFR3, RBI, HRAS, KRAS, TP53, TSC1, FGFR3

BRCA, BRCA 2, BARD1, BRIP1, CHEK2, MRE1 1A, NBN, Breast and Ovarian PALB2, PTEN, RAD50, RAD50, RAD51C, RAD51D, PPMDD, TP53, BRIP1 , RAD54L, SLC22A1 L, PIK3CA, RB1CC1 ,

Cervical FGFR3

PT53, STK1 , PTEN, BMPR1A, SMAD, MLH l, MSH2, Colon and Rectal MSH6, PMS, EPCAM, AKT1, APC, MYH, PTPRJ, AXIN2

Endonietrial/Uterine MLH , MSH2, MSH6, PMS, EPCAM

Esophageal DLEC1, TGFBR2, RNF6, LZT1S1, WWOX

Hepatocellular carcinoma PDGFRL, CTNNB1, TP53, MET, CASP8, PIK3CA

Renal VHL, PBRMQ, BAP1, SETD2, HIF1- KRAS, EGFR, ALK,BRAF, ERBB2, FLCN, DIRC2, RNF139, Lung OGG1, PRCC, TFE, MET, PPP2R1B, RASSF1, SLC22A1L BRAF, CDKA, CDKN2A, CDKN2B, CDKND, MCIR, TERT, Melanoma ATF1, CREB1, EWSR1

Non-Hodgkin Lymphoma CASP 10, EGFR, IRF1, PIK3CA

Osteosarcoma CKEK2, LOJ18CR1, RBI

Ovarian PRKN, AKT1

KRAS, BRCA2, CDKN2A, MANF, PALB2, SMAD4, TP53, Pancreatic IPF1 MLHl, MSH2, MSH6, and PMS2, BRCA 1, HOXB13, CHEK2, Prostate ELAC2, EPHB2, SDR5A2, PRKAR1A, PMC1 Papillary and Follicular BRAF, NARAS, ERCI, FOXEl, GOLGA5, NCOA4, NKX2-1 , Thyroid PMC1, ET, TFG, TPR, TREM24, TRIM27, TRIM33

Erwing Sarcoma ERG, ETV 1, ETV4, EWSR 1, FLI 1 BRC, AMCR2, GMPS, JAK2, AF10, ARFGEF12, CEBPA, Leukemia FLT3, KIT, LPP, MLF1, NPM1, NSD1, NUP214, PICALM, RUNXl, SH3GL1, WHSC1L1, ETV6, RARA, BCR, ARHGAP26, NFl, PTPN1 1, GATAl

[0806] Any of the following cancer associated genes provided in Table VII- 16 can be targeted. [0807] Table VII- 16 Exemplary Target Genes Associated With Cancer:

Table V - 6:

ABL 1, ABL2, ACSL3, AF1 5Q 14, AF1 Q, AF3p2 1, AF5q3 1, AKAP9, A T 1, AKT2, ALDH2, AL , AL01 7, A C, ARHGEF 12, ARHH, ARID1 A, ARID2, ARNT, ASPSCR 1, ASXL1 , ATF1 , ATIC, ATM, ATRX, AXIN 1, BAP1 , BCL10, BCL1 1A, BCL1 B, BCL2, BCL3, BCL5, BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPR IA, BRAF, BRCA l , BRCA2, BRD3, BRD4, BRIP I,

BTG 1, BUB 1B, C 12ori9, C 15ort2 1, C 15orf55, C 16orl75, C2orf44, CAMTA 1, CANT1 , CARD1 1,

CARS, CBFA2T 1, CBFA2T3, C.BFB, CBL, CBLB, CBLC, CCDC6, CCNB 1IP1 , CCND1 , CCND2, CCND3, CCNE 1, CD273, CD274, CD74, CD79A, CD79B, CDH 1, CDH 11, CDK1 2, CDK4, CDK6, CDKN2A, CDKN2a(p l4), CD N2C, CDX2, CEBPA, CEPl , CHCHD7, CHEK2, CHIC2, CHNl , CIC,

A, CLTC, CLTCL 1, CMK R 1, CNOT3, COL1 A , COPEB, COX6C, CREB 1, CREB3L 1,

CREB3L2, CREBBP, CRLF2, CRTC3, CTNNB 1, CYLD, D 0S1 70, DAXX, DDB2, DDIT3, DDX 10, DDX5, DDX6, DEK, D1CER 1, DNM2, DNMT3A, DUX4, EBFI , ECT2L, EGFR, E1F4A2, ELF4, ELK4, ELKS, ELL, ELN, EML4, EP300, EPS 15, ERBB2, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ETV l , ETV4, ETV , ETV6, EVIl , EWSR 1, EXTl , EXT2, EZH2, EZR, FACL6, FAM22A, FAM22B, FAM46C, 1ANCA, EANCC, FANCD2, FANCE, FANCF, FANCG, FBXOl 1, FBXW7, FCGR2B,

FEV, FGFR 1, FGFR OP, FGFR2, FGFR3, F , F T, FIP1 L 1, FL , FLJ27352, FLT3, FNBP1 , FOXL2, FOXO IA, FOX03A, FOXP 1, FSTL3, FUBP1 , FUS, FVT 1, GAS7, GATA 1, GATA2, GATA3, GMPS, GNA 11, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GRAF, H3F3A,

ICMOGT- , IEAB, HERPUD1 , IIEY1 , IIIPl , HIST1 IT3B, IIIST1 I I4I, IILF, HLXB9, HMGA 1,

HMGA2, HNRNPA2B I , HOOK3, HOXA1 1, HOXA1 3, HOXA9, HOXC 11, HOXC1 3, HOXD 11,

HOXD 13, HRAS, IIRPT2, HSPCA, HSPCB, IDH l , IDH2, IGH@, IGK@, IGL@, IKZFl , IL2, L 2 1R, IL6ST, IL7R, IRF4, IRTA 1, ITK, JAK1 , JAK2, JAK3, JAZF1 , JUN, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KIAA 1549, KIF5B, KIT, KLF4, KLK2, KRAS, KTN 1, LAF4, LASPl , LCK, LCP1 , LCX, LHFP, LIFR, LMO l , LM02, LPP, LRIG3, LYL1 , MADH4, MAF, MAFB, MALT1 , MAML2, MAP2K MAP2K2, ΜλΡ 2Κ4, MAX, MDM2, MDM4, MDS 1, MDS2, MECTl , MED 12, MEN 1, MET, MITF, MKL1 , MLF1 , ML Il , MLL, MLL2, MLL3, MLLT1 , MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1 , MUC 1, MUTYH, MYB, MYC, MYCL 1, MYCN, MYD88, MYH 1, MYH9, MYST4, NACA, NBS 1, NCOA 1, NCOA2, NCOA4, NDRG 1, N 1, NF2, NFE2L2, NFIB, NFKB2, NIN, NKX2- 1, NONO, NOTCH , NOTCH2, Table VII-16:

NPMl, NR4A3, NRAS, NSDl, NT5C2, NTRKl, NTRK3, NUMAl, NUP214, NUP98, OLIG2, OMD,

P2RY8, PAFAH1B2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PIIF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG 1, PML, PMS1, PMS2, PMX1, PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A,

PRCC, PRDM 1, PRDM16, PRF1 , PRKAR1 A, PRO1073, PSIP2, PTCH, PTEN, PTP , RAB5EP, RACl, RAD51L1, RAFl, RALGDS, RANBP17, RAPIGDSI, RARA, RBI, RBM15, RECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB, SDHC, SDHD, SEPT6, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMARCA4, SMARCB1, SMARCE1, SMO, SOCS1, SOX2,

SRGAP3, SRSF2, SSI , SS18L1, SSH3BP1, SSX1, SSX2, SSX4, STAT3, STK1 , STL, SUFU, SIJZ12, SYK, TAF15, TALI, TAL2, TCEA1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG, TFPT, TFRC, THRAP3, TIF1, TLX1, TL 3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TNFRSF6, TOPI, TP53, TPM3, TPM4, TPR, TRA@, TRAF7, TRB@, TRD@, TRIM27, TRIM33, TRIP1 1, TSC1 , TSC2, TSHR, TTL, U2AF1, USP6, VHL, VT A, WAS, W S , WHSC1L1, WIF1, WRN, WT1, WTX, WWTR1, XPA, XPC, XPOl, YWHAE, ZNF145, ZNF1 8, ZNF278, ZNF33 1, ZNF384, ZNF52 1, ZNF9, or ZRSR2

[0808] Exemplary pathways and genes associated with energy metabolism are provided in Table VII-17. Exemplary metabolic targets disclosed herein may be modulated using CRISPR/Cas9 as described herein. Modulation may be used to knockdown a gene of interest, correct a defect or mutation in the gene, or to activate a gene of interest.

Table VII-17 GL, gastric lipase Knock down PL, pancreatic lipase Knock down sPLA2, secretory phospholipase A2 Knock down ACC, acetyl-CoA carboxylase Knock down CPT, carnitine palmitoyl transferase Knock down FAS, fatty-acid synthase Knock down MTP, microsomal triglyceride-transfer protein Knock down Insulin receptor Correct defects or activate SU receptor/K+ ATP channel Activate with mutation a-glucosidase Knock down PPARy Activate with mutation Glycogen phosphorylase Knock down Fructose- 1, 6-bisphosphatase Knock down glucose-6-phosphatase Knock down PTP-1B Knock down SHIP-2 Knock down GSK-3 Knock down l B kinase Knock down PKCq Knock down GLP1R Correct mutation GIPR Correct mutation GPR40 Correct mutation GPR1 9 Correct mutation GPR41 Correct mutation GPR43 Correct mutation GPR120 Correct mutation GCGR Correct mutation PAC1 Correct mutation VPAC2 Correct mutation Y l Knock down GHSR Knock down CC AR Correct mutation b2 Correct mutation a2 Knock down MT1 Knock down M3 Correct mutation CB 1 Knock down P2Y Correct mutation H3 Inhibit MCH-R ] Coirect mutation MCH-R2 Correct mutation Ghrelin R Inhibit FASN Inhibit Bombesin-R3 Inhibit CCK-A Receptor Correct mutation Seratonin System Correct mutation CBI Cannabinoid Receptors Inhibit Dopaminergic System Correct mutation Enterostatin Mutate to super agonist CNTF Mutate to super agonist CNTF-R Correct mutation SOCS-3 Knock down 46a Knock down PrPP Receptors Correct mutation Amylin Mutate to super agonist CRH System Mutate to super agonist Galanin Receptors Knock down Orexin Receptors Knock down Noradrenalin System Mutate to super agonist CART Mutate to super agonist FATP4 Knock down Pancreatic Lipase Knock down ACRP30 Super agonist mutations Thyroid Hormone Correct mutation B-3 Adrenergic Receptor Correct mutation UCPs Upregulate PTP-1B Knock down MC3 Correct mutation ACC2 Knock down Perilipin Knock down HMGIC Knock down l lBHSD-1 Knock down Glucagon R Knock down Glucocoricoid R Knock down 1lbeta-HSD I Knock down PGC-1 Correct mutation DPPP-rV Knock down GLP Mutate to super agonist GIP Mutate to super agonist GLP-IR Correct mutation AMP Kinase Correct mutation IKK-b Knock down PPARa/g Knock down INS-R Knock down SGLT Knock down- a-glucosidase Knock down HMGCR Knock down PCSK9 Knock down ApoB-100 Knock down Leptin Mutate to super agonist Leptin Receptor Mutate to constitutively active receptor MC4R Mutate to constitutively active receptor VOMC Mutate MSH region to super agonist AGRP Knock down rVPY Receptors Introduce constitutively active mutations 5HT2C Introduce constitutively active mutations GLP-1 Mutate to super agonist GLP-1 Receptor Mutate to constitutively active receptor

[0809] In an embodiment, the pathways and genes described herein, e.g., in Table VII- 7, are also associated with diabetes, obesity, and/or cholesterol and lipids.

[0810] Exemplary pathways and genes associated with the cell cycle are provided in Table VII- 18.

Table VII-18 MRE1 1 MLH1 FasR NBS 1 MSH6 Trail-L RAD50 MSH2 Trail-R 53BP1 RFC TNF-Ct P53 PCNA TNF-R1 CHKE MSH3 FADD E2F1 MutS homolog TRADD PML MutL homolog R1P1 FANCD2 Exonuclease MyD88 SMC1 DNA Polymerase delta IRAK BLM1 (POLDl, POLD2,POLD3, NIL BRCA 1 and POLD4 -genes IKK H2AX encoding subunits) NF- Κβ ATR Topoisomerase 1 ΙκΒα RPA Topoisomerase 2 IAP ATRIP RNAseHl Caspase 3 RAD9 L ase 1 Caspase 6 RAD1 DNA polymerase 1 Caspase 7 HUS DNA polymerase 3 Caspase 8 RAD 17 Primase Caspase 10 RFC Helicase HDAC1 CHK1 Single-strand binding HDAC2 TL 1 proteins Cytochrome C CDC25 Bxl-xL STAT3 STAT5 DFF45 Vcl-2 ENDO-G PI3K Akt Calpain Bad Bax

Ubiqiiitin-mediated proteolysis Hypoxia Cell Proliferation HIF-loc APK E l HERC1 TRAF6 HIF- Ιβ MAPKK E2 UBE2Q MEKK1 Refl MAPKKK E3 UBE2R COP! HSP90 c-Met UBLE1A UBE2S PIFH2 VEGF HGF UBLE1B UBE2U cIAP PAS ER S 1/2 UBLEIC UBE2W PIAS ARNT ATK UBE2A UBE2Z SYVN VHL PKCs UBE2B AFCLLCN NHLRC1 HLF Paxilin UBE2C UBE1 AIRE EPF FAK UBE2A E6AP MGRN1 VDU2 Adducin UBE2E UBE3B BRCA1 SUMORESUME PYK1 UBE2F Smurf FANCL SENP1 RB UBE2G1 Itch MIDI Calcineurin A RBI UBE2G2 HERC2 Cdc20 RACK1 Raf-1 UBE2I HERC3 Cdhl PTB A-Raf UBE2J1 HERC4 Apcl Hur B-raf UBE2J2 UBE4A Apc2 PHD2 ME 1/2 UBE2L3 UBE4B Apc3 SSAT2 ERK1/2 UBE2L6 CHIP Apc4 SSAT1 Ets UBE2M CYC4 Apc5 GSK3 E UBE2N PPR19 Apc6 CBP SAP1 UBE20 UIP5 Apc7 FOX04 cPLA2 WWPI Mdm2 Apc8 FlH-1 WWP2 Parkin Apc9 TRIP 12 Trim32 Ape 10 NEED4 Trim37 Ape 11 ARF-BP1 SIAH-1 Ape 12 EDD1 PML Cell survival Cell cycle arrest S D P SMAD5 BAX SAMD8 MDR LEF1 DRAIL IGFBP3 TCF3 GADD45 TCF4 P300 HAT1 PI3 Akt GF1

[0811] Exemplary cell cycle genes characterized by their function are provided in Table VII-19. Table V - 9

[0812] Exemplary pathways and genes associated with the angiogenesis are described provided in Table V -20.

Table VII-20 VEGF VEGFR2 SHC E2F7 VEGFB VEGFR3 PI3 VEGFC Nrpl PIP3 VEGFD IP3 DAG GRB2 SOS Akt P B PKC Ras RAF1 DAG eNOS NO ERK1 ER 2 cPLA2 ME 1 MEK2

[0813] Exemplary pathways and genes associated with the mitochondrial function are provided in Table VII-25.

Table VII-25 Malate dehydrogenase Aminotransferase Hydratase Deacylase Dehydrogenase Carboxylase Mutase

Fatty acid oxidation Leucine Oxidation Isoleucine disorders (enzyme Pathway oxidation pathway deficiencies) Aminotransferase Aminotransferase OCTN2 Branched chain Branched chain FATP1-6 aminotransferase 2, aminotransferase 2, CPT-1 mitochondrial mitochondrial CACT Isobutytyl-CoA 2-methylbutytyl-CoA CPT-II dehydrogenase Dehydrogenase SCAD (Branched Chain (Branched Chain MCAD Keto Acid Keto Acid VLCAD Dehydrogase Dehydrogenase ETF-DH Complex) Complex) Alpha-ETF Hydratase Hydratase Beta-ETF HMG-CoA lyase 2-methyl-3-OH- SCHAD butyryl-CoA LCHAD dehydrogenase MTP 3-Oxothiolase LKAT DECR1 HMGCS2 HMGCL Additional mitochondrial genes and related diseases caused by mutations Mt-NDl Leber's hereditary optic neuropathy Mt-ND4 Leber's hereditary optic neuropathy Mt-ND6 Leber's hereditary optic neuropathy OPA1 Autosomal dominant optic atrophy C T2A Charcot-Marie-Toothhereditary neuropathy type 2A mt-T Myoclonic epilepsy with ragged red fibres Mitochondrial Related diseases Respiratory chain genes NADH CoQ Alpers, Alzheimer's, Parkinsonism, Cardiomyopathy, Deficiency (Barth Reductase and/or Lethal Infantile), Encephalopathy, Infantile CNS, Leber's, Leigh, Longevity, MELAS, MERRF, Myopathy ± CNS, PEO, Spinal cord disorders Succinate-CoQ Keams-Sayre, Leigh's, Myopathy (e.g., Infantile ± CNS), Paraganglioma, Reductase Pheochromocytoma CoQ-Cytochrome C Cardiomyopathy, Fatal infantile, GRACILE, Leber's, Myopathy (e.g., ± Reductase CNS, PEO) Cytochrome C Alper's, Ataxia, Deafness, Leber's, Leigh's, Myopathy (e.g., Infantile (e.g., Oxidase Fatal, Benign), Adult), Rhabdomyolysis, PEO, KSS, MNGEE, MERRF, MELAS

ATP Synthase Cardiomyopathy, Encephalopathy, Leber's, Leigh, Multisystem, NARP Complex I (NADH-Ubiquinone Oxidoreductase) Nuclear encoded Mitochondral DNA Supernumerary Subunits involved proteins encoded proteins subunits in regulation of NDUFAB1 (SDAP): Complext I activity NDUFS 1: Childhood NDl Carrier of fatty acid NDUFS4 (AQDQ) encephalopathy; Most chain common Complex 1 ND2 Functions: mutations (3%) NDUFA1 (MWFE) Increased Complex ND3 I activity with NDUFS2: Primarily expressed phosphorylation Cardiomyopathy + ND4 in heart & skeletal Encephalomyopathy muscle Disorders: ND4L Multisystem NDUFS3: Leigh Disorders: childhood ND5 Encephalopathies encephalopathy NDUFS7: Leigh with Complex I ND6 NDUFA2: deficiency; Leigh NDUFS8: Leigh Encephalopathy & syndrome Cardiomyopathy NDUFV1 : Childhood encephalopathy NDUFA9: Leigh syndrome NDUFV2: Encephalopathy + NDUFA10: Leigh Cardiomyopathy syndrome

ELAC2: NDUFA1 Cardiomyopathy, Hypertrophic Disorder: Encephalopathy & Cardiomyopathy

NDUFA12: Leigh syndrome

NDUFB9: Hypotonia

NDUFS6: Lethal Infantile Mitochondrial Disease

Proteins involved in Other Complex I assembly NDUFA 3 : Thyroid • NDUFAF1 : carcinoma (Hurthle Cardiomyopathy + cell) Encephalomyopathy • NDUFAF2 NDUFB3: Severe (NDUFA12L): lethal mitochondrial Childhood complex I deficiency encephalopathy; Usually null MTHFR deficiency mutations - • NDUFAF3: Lethal MGME1: PEO + neonatal Myopathy encephalopathy • NDUFAF4: Encephalopathy • C6ORF66: Encephalopathy • C8orf38: Leigh syndrome • C20orf7: Lethal neonatal • NUBPL: Encephalomyopathy • ACAD9: Fatigue & Exercise intolerance; Most missense mutations • FOXRED1: Leigh syndrome • Ecsit • A1F (A1FM1 ; - PDCD8) • Indl

Complex (NADH-lJbit|uinoiu' Oxidoroductase) Flavoprotein: FAD (SDHA; Fp) • Mutations cause Leigh syndrome with Complex II deficiency • Late onset neurodegenerative disorder)

Iron-Sulfur protein: SDHB (Ip) 0 Mutations cause Reduced tumor suppression 0 Neoplasms: Pheochromocytoma & Paraganglioma

SDHC ; SDHD (cytochrome C subunits) - o mutations lead to paraganglioma

Complex HI (Cytochrome reductase) Cytochrome c l (CYC1) Rieske FeS protein (UQCRFS1) Ubiquinol-cytochrome c reductase core May mediate formation of complex between protein I (UQCRC1; QCR; Subunit 1) cytochromes c and c Ubiquinol-cytochrome c reductase core Required for assembly of complex III protein II (UQCRC2; QCR2; Subunit 2) UQCRH (Subunit 6) May mediate formation of complex between cytochromes c and c l Ubiquinone-binding protein (UQBC; Redox-linked proton pumping UQPC; UQCRB; UQBP; Subunit 7) UOCRO (Subunit 8) Binds to ubiquinone Ubiquinol-cytochrome C reductase Interacts with cytochrome c l complex, 7.2-KD Subunit (UCRC; UQCR 10; Subunit 9) UQCR (UQCR 11; Subunit 10) function as iron-sulfur protein binding factor Cleavage product of UQCRFS1 (Cytochrome b-cl complex subunit 11) Inner membrane proteins and related disorders ABCB7: Ataxia + Anemia ACADVL: Myopathy ADC 3: SACR9 AGK: Sengers ATP5A1 : Encephalopathy, neonatal ATP5E: Retardation + Neuropathy BRP44L: Encephalopathy c l2orf62: Encephalocardiomyopathy Cardiolipin: Barth COX4I2: Pancreas + Anemia COX6B 1: Encephalomyopathy CPT2: Myopathy C AT: Encephalomyopathy CYC1 : Hyperglycemia & Encephalopathy CYCS CYP1 1A1 CYP1 1B1 CYP1 1B2 CYP24A1 CYP27A1 : Cerebrotendinous Xanthomatosis CYP27B1 DHODH DNAJC19: Cardiac + Ataxia FASTKD2: Encephalomyopathy GPD2

HADHA: Multisystem; Myopathy HADHB: Encephalomyopathy HCCS: MIDAS L2HGDH: Encephalopathy MMAA MPV17: Hepatocerebral NDUFAl : Encephalopathy NDUFA2: Leigh + Cardiac NDUFA4: Leigh NDUFA9: Leigh NDUFA10: Leigh NDUFAl 1: Encephalocardiomyopathy NDUFA12: Leigh NDUFAl 3 NDUFB3: Lethal infantile NDUFB9: Encephalopathy NDUFV1 : Encephalopathy NDUFV2: Encephalopathy + Cardiac NDUFSl: Leukodystrophy NDUFS2: Encephalopathy + Cardiac NDUFS3: Dystonia NDUFS4: Encephalopathy NDUFS6: Lethal infantile NDUFS7: Encephalopathy NDUFS8: CNS + Cardiac OPA 1: Optic atrophy

OPA3: Optic atrophy PDSS1 : Coenzyme Q10 deficiency SDHA: Leigh; Cardiac; Paraganglioma SDHB: Paraganglioma SDHC: Paraganglioma SDHD: Paraganglioma SLC25A earners SLC25A1: Epileptic encephalopathy SLC25A3: Cardiac; Exercise intolerance SLC25A4: PEOA2 SLC25A12: Hypomyelination SLC25A13: Citrullinemia SLC25A15: HHH SLC25A : Microcephaly SLC25A20: Encephalocardiomyopathy SLC25A22: Myoclonic epilepsy SLC25A38: Anemia Paraplegin: SPG7 TIMM8A: Dea '-Dystonia-Dementia UCP1 UCP2 UCP3 UQCRB: Hypoglycemia, Hepatic UQCRC2: Episodic metabolic encephalopathy UQCRQ: Encephalopathy

[0814] Pathways and genes associated with DNA damage and genomic instability include the following methyl transferases, histone methylation, helicase activity, nucleotide excision repair, recombinational repair, or mismatch repair provided in Table VII-21. See also Table VI-22.

Table VII-21 53BP1 BLM MBD2 DNA ligase 4 MDC1 H2AX XLF SMC1 53BP1 Rad50 P53 P53 Artemis Rad27 TdT Base-Excision repair Nucleotide-Excision Homologous Mismatch repair APE1 Repair Recombination PMS2 APE2 UvrA RecA MLH1 NEIL1 UvrB SSB MSH6 NEIL2 UvrC Mrel l MSH2 NEIL3 XPC Rad50 RFC XRCC1 Rad23B Nbsl PCNA PNKP CEN2 CtIP MSH3 Tdpl DDB1 RPA MutS APTX XPE Rad51 MutL DNA polymerase β CSA, Rad52 Exonuclease DNA polymerase δ CSB Rad54 Topoisomerase 1 DNA polymerase ' s TFT1H BRCA1 Topoisomerase 2 PCNA XPB BRCA2 RNAseHl FEN1 XPD Exol Ligase 1 RFC XPA BLM DNA polymerase 1 PAR 1 RPA Topllla DNA polymerase 3 Lig XPG GEN 1· Primase Lig3 ERCC1 Yenl Helicase UNG XPF Slxl SSBs MUTY DNA polymerase δ Slx4 SMUG DNA polymerase ε Mus8 MBD4 E e l Dssl Histone Methylation ASH 1L SETD4 DQT1 L SETD5 EHMT1 SETD6 EHMT2 SETD7 EZH 1 SETD8 EZH2 SETD9 MLL SETDB1 MLL2 SETDB2 MLL3 SETMAR MLL4 SMYD1 MLL5 SMYD2 NSD1 SMYD3 PRDM2 SMYD4 SET SMYD5 SETBP1 SUV39H1 SETD1A SUV39H2 SETD1B SUV420H1 SETD2 SUV420H2 SETD3 Table VII-22

Selected Transcript ion FactorsTranscriptionfactors

NIKX2-5 Cardiac malformations and atrioventricular conduction abnormalities MECP2 Rett syndrome HNF1 through Mature onset diabetes of the young HNF6 (MODY), hepatic adenomas and renal cysts FOXP2 Developmental verbal dyspraxia FOXP3 Autoimmune diseases NOTCH 1 Aortic valve abnormalities MEF2A Coronary artery disease CRX Dominant cone-rod dystrophy FOCX2 Lymphedema-distichiasis NF-KB Autoimmune arthritis, asthma, septic Activation shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS NF-KB Inhibition Apoptosis, inappropriate immune cell development, and delayed cell growth NARA2 Parkinson disease LHX3 Pituitary disease GAT4 Congenital heart defects P53, APC Cancer CTCF Epigenetics and cell growth regulation EGR2 Congenital hypomyelinating neuropathy (CHN) and Charcot-Marie- Tooth type 1 (CMT1) STATfamily Cancer and immunosuppression NF-A Tfamily Cancer and inflammation . AP-1 family Cancer and inflammation

[0815] A gene including receptors and ionophores relevant to pain in this table can be targeted, by editing or payload delivery. Pathways and genes associated with pain are described herein, e.g., include the following those in Table VII-24.

Table VII-24 nociceptive central 5HT3 central inhibition antiemetic nociceptive central 5HT4 central inhibition gastroproknetics nociceptive central 5HT5A central inhibition nociceptive central 5HT5B central inhibition nociceptive central 5HT6 central inhibition antidepressant (antagonists and agonists), anxiolytic (antagonists and agonists), nootropic (antagonists), anorectic (antagonists) nociceptive central 5HT7 central inhibition antidepressant (antagonists), anxiolytics (antagonists), nootropic (antagonists) nociceptive central CB1 central inhibition nociceptive central GABA central inhibition nociceptive central GABAA-$ central inhibition nociceptive central GABAB-R central inhibition nociceptive central Glucine-R central inhibition nociceptive central NE central inhibition nociceptive central Opiod central inhibition receptors nociceptive central c-fos gene expression nociceptive central c-jun gene expression nociceptive central CREB gene expression nociceptive central DREAM gene expression nociceptive peripheral + channel membrane excitability of primary afferents nociceptive peripheral Navl.8 membrane excitability of primary afferents nociceptive peripheral Navl .9 membrane excitability of primary afferents nociceptive peripheral CaMKIV peripheral sensitization nociceptive peripheral COX2 peripheral sensitization nociceptive peripheral cPLA2 peripheral sensitization nociceptive peripheral EP1 peripheral sensitization nociceptive peripheral EP3 peripheral sensitization nociceptive peripheral EP4 peripheral sensitization nociceptive peripheral ER 1/2 peripheral sensitization nociceptive peripheral L- 1beta peripheral sensitization nociceptive peripheral JNK peripheral sensitization nociceptive peripheral Navl.8 peripheral sensitization nociceptive peripheral NGF peripheral sensitization nociceptive peripheral p38 peripheral sensitization nociceptive peripheral PKA peripheral sensitization nociceptive peripheral P C peripheral isoforms sensitization nociceptive peripheral TNFalpha peripheral sensitization nociceptive peripheral TrkA peripheral sensitization nociceptive peripheral TRPV1 peripheral sensitization nociceptive central AMPA/kain postsynaptic ate-R transmission nociceptive central + channels postsynaptic transmission nociceptive central mGlu-$ postsynaptic transmission nociceptive central Navl .3 postsynaptic transmission nociceptive central N 1 postsynaptic transmission nociceptive central NMDA-R postsynaptic transmission nociceptive peripheral Adenosine- presynaptic R transmission nociceptive peripheral mGluR presynaptic transmission nociceptive peripheral VGCC presynaptic transmission nociceptive central ERK signal transduction nociceptive central JNK signal transduction nociceptive central p38 signal transduction nociceptive central PKA signal transduction nociceptive central PKC signal isoforms transduction nociceptive peripheral ASIC transduction nociceptive peripheral B 1 transduction nociceptive peripheral BK2 transduction nociceptive peripheral DRASIC transduction nociceptive peripheral MDEG transduction nociceptive peripheral P2X3 transduction nociceptive peripheral TREK-1 transduction nociceptive peripheral TRPM8 transduction nociceptive peripheral TRPV1 transduction nociceptive peripheral TRPV2 transduction nociceptive peripheral TRPV3 transduction neuropathic pain Inflammatory histamine pain Inflammatory ATP pain Inflammatory bradykinin pain Inflammatory CB2 pain Inflammatory Endothelins pain Inflammatory H+ pain Inflammatory Interleukins pain Inflammatory NGF pain Inflammatory prostaglandi pain ns Inflammatory serotonin pain Inflammatory TNFalpha pain

VII I. TARGETS: DISORDERS ASSOCIATED WITH DISEASE CAUSING ORGANISMS

[081 6] Cas9 molecules, typically eiCas9 molecules or eaCas9 molecules, and gRNA molecules, e.g., an eiCas9 molecule/gRNA molecule complex, e.g., an eaCas9 moIecule/gRNA molecule complex, can be used to treat or control diseases associated with disease causing organisms, e.g., to treat infectious diseases. In an embodiment, the infectious disease is treated by editing (e.g., correcting) one or more target genes, e.g., of the organism or of the subject. In other embodiments, the infectious disease is treated by delivering one or more payloads (e.g., as described herein) to the cell of a disease causing organism or to an infected cell of the subject, e.g., to a target gene. In some embodiments, the target gene is in the infectious pathogen. Exemplary infectious pathogens include, e.g., viruses, bacteria, fungi, protozoa, or mutlicellular parasites.

[081 7] In other embodiments, the target gene is in the host cell. For example, modulation of a target gene in the host cell can result in resistance to the infectious pathogen. Host genes involved in any stage of the life cycle of the infectious pathogen (e.g., entry, replication, latency) can be modulated. In an embodiment, the target gene encodes a cellular receptor or co-receptor for the infectious pathogen. In an embodiment, the infectious pathogen is a virus, e.g., a virus described herein, e.g., HIV. In an embodiment, the target gene encodes a co-receptor for HIV, e.g., CCR5 or CXCR4. [0818] Exemplary infectious diseases that can be treated by the molecules and methods described herein, include, e.g., AIDS, Hepatitis A, Hepatitis B, Hepatitis C, Herpes simplex, HPV infection, or Influenza.

[0819] Exemplary targets are provided in Table VID-1. The disease and causative organism are provided.

Table VIIl-1

DISK/VSR SOURCE OF DISEASE Acinetobacler infcclions Acinetobacler baumannii

Actinomyces israelii. Actinomyces gerencseriae and Actinomycosis Propionibacterium propionicus

African sleeping sickness (African trypanosomiasis) Trypanosoma brucei

AIDS (Acquired immunodeficiency syndrome) HIV (Human immunodeficiency virus) Amebiasis Entamoeba histolytica Anaplasmosis Anaplasma genus Anthrax Bacillus anthracis

Arcanobacterium haemolyticum infection Arcanobacterium haemolyticum Argentine hemorrhagic fever Junin virus Ascariasis Ascaris lumbricoides Aspergillosis Aspergillus genus Astrovirus infection Astroviridae family Babesiosis Babesia genus Bacillus cereus infection Bacillus cereus Bacterial pneumonia multiple bacteria Bacterial vaginosis (BV) multiple bacteria Bacleroides infection Bacteroides genus Balantidiasis Balantidium coli Bavlisasearis infection Baylisascaris genus B virus infection BK virus Black picdra Piedraia hortae Blaslocystis hominis infection Blastocystis hominis Blaslomvcosis Blastomyces dermalilidis Bolivian hemorrhagic fever Machupo virus Borrelia infection Borrelia genus Clostridium botulinum; Note: Botulism is not an infection by Clostridium botulinum but caused by the intake of Botulism (and Infant botulism) botulinum toxin. Brazilian hemorrhagic fever Sabia Brucellosis Brucella genus Bubonic plague the bacterial family Enterobacteriaceae

usually Burkholderia cepacia and other Burkholderia Burkholderia infection species Buruli ulcer Mycobacterium ulcerans

Calicivirus infection (Norovirus and Sapovinis) Caliciviridae family Campylobaclcriosis Campylobacter genus

Candidiasis (Moniliasis; Thrush) usually Candida albicans and other Candida species Cat-scratch disease Bartonella henselae

Cellulitis usually Group A Streptococcus and Staphylococcus

Chagas Disease (American trypanosomiasis) Trypanosoma cruzi Chancroid Haemophilus ducreyi Chickenpox Varicella zoster virus (VZV) Chlamydia Chlamydia trachomatis

Chlamydophila pneumoniae infection (Taiwan acute respiratory agent or TWA ) Chlamydophila pneumoniae Cholera Vibrio cholerae Chromoblastomvcosis usually Fonsccaca pedrosoi Clonorchiasis Clonorchis sinensis Clostridium difficile infection Clostridium difficile Coccidioidomycosis Coccidioides immitis and Coccidioidcs posadasii Colorado tick fever (CTF) Colorado tick fever virus (CTFV)

Common cold (Acute viral rhinopharyngitis; Acute coryza) usually rhinoviruses and coronaviruses. Creutzfeldt-Jakob disease (CJD) PRNP

Crimean-Congo hemorrhagic fever (CCHF) Crimean-Congo hemorrhagic fever virus Cryptococcosis Cryptococcus neoformans Cryptosporidiosis Cryptosporidium genus

Cutaneous larva migrans (CLM) usually Ancylostoma braziliense; multiple other parasites Cyclosporiasis Cyclospora cayetanensis Cysticercosis Taenia solium Haemophilus influenzae infection Haemophilus influenzae Enteroviruses, mainly Coxsackie Λ virus and Enterovirus Hand, loot and mouth disease (H M ) 7 1 (EV7 I)

Hantavirus Pulmonary Syndrome (HPS) Sin Nombre virus Helicobacter pylori infection Helicobacter pylori Hemolytic-uremic syndrome (HUS) Escherichia coli 0157:H7, O l 11 and O104:H4

Hemorrhagic fever with renal syndrome (HFRS) Bunyaviridae family Hepatitis A Hepatitis A Virus Hepatitis B Hepatitis B Virus Hepatitis C Hepatitis C Virus Hepatitis D Hepatitis D Virus Hepatitis E Hepatitis E Vi s

Herpes simplex Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) Histoplasmosis Histoplasma capsulatum

Hookworm infection Ancylosloma duodenale and Necator americanus Human bocavinis infection Human bocavirus (HBoV) Human ewingii ehrlichiosis Ehrlichia ewingii

Human granulocytic anaplasmosis (HGA) Anaplasma phagocylophilum

Human metapneumovirus infection Human metapneumovirus (hMPV) Human monocytic ehrlichiosis Ehrlichia chaffeensis

Human papillomavirus (I IPV) infection Human papillomavirus (HPV)

Human parainfluenza virus infection Human parainfluenza viruses (HPIV) Hymenolepiasis Hymenolepis nana and Hymenolepis diminula

Epstein-Barr Virus Infectious Mononucleosis (Mono) Epstein-Barr Virus (EBV) Influenza (flu) Orthomyxoviridae family Isosporiasis Isospora belli

Kawasaki disease unknown; evidence supports that it is infectious Keratitis multiple Kingella kingae infection Kingella kingae Kuru PRNP Lassa fever Lassa virus

Legionellosis (Legionnaires' disease) Legionella pneumophila

Pelvic inflammatory disease (PID) multiple Pertussis (Whooping cough) Bordetella pertussis Plague Yersinia pestis Pneumococcal infection Streptococcus pneumoniae Pneumocystis pneumonia (PCP) Pneumocystis jirovecii Pneumonia multiple Poliomyelitis Poliovirus Prevotella infection Prevotella genus

Primary amoebic meningoencephalitis (PAM) usually Naegleria fovvleri

Progressive multifocal leukoencephalopathy JC virus Psittacosis Chlamvdophila psittaci Q fever Coxiella burnetii Rabies Rabies virus

Rat-bite fever Slreptobacillus moniliformis and Spirillum minus

Respiratory syncytial virus infection Respiratory syncytial virus (RSV) Rhinosporidiosis Rhinosporidium seeberi Rhinovinis infection Rhinovirus Rickettsial infection Rickettsia genus Rickettsialpox Rickettsia akari Rift Valley fever (RVF) Rift Valley fever virus

Rocky Mountain spotted fever (RMSF) Rickettsia rickettsii Rotavirus infection Rotavirus Rubella Rubella virus Salmonellosis Salmonella genus

SARS (Severe Acute Respiratory Syndrome) SARS coronavirus Scabies Sarcoptes scabiei Schistosomiasis Schistosoma genus Sepsis multiple Shigellosis (Bacillary dysentery) Shigella genus Shingles (Herpes zoster) Varicella zoster virus (VZV) Smallpox (Variola) Variola major or Variola minor Sporotrichosis Sporothrix schenckii Staphylococcal food poisoning Staphylococcus genus Staphylococcal infection Staphylococcus genus Strongyloidiasis Strongyloides stercoralis Yersiniosis Yersinia enterocolilica Yellow lever Yellow fever virus

Mucorales order (Mucormycosis) and Entomophthorales Zygomycosis order (Entomophthoramycosis)

AIDS/HIV

HIV genomic structural elements

[0820] Long terminal repeat (LTR) refers to the DNA sequence flanking the genome of integrated proviruses. It contains important regulatory regions, especially those for transcription initiation and polyadenylation.

[0821] Target sequence (TAR) for viral transactivation, the binding site for Tat protein and for cellular proteins; consists of approximately the first 45 nucleotides of the viral mRNAs in HIV-1 (or the first 100 nucleotides in HIV-2 and SIV.) TAR RNA forms a hairpin stem-loop structure with a side bulge; the bulge is necessary for Tat binding and function.

[0822] Rev responsive element (RPE) refers to an RNA element encoded within the env region of HIV-1 . It consists of approximately 200 nucleotides (positions 7327 to 7530 from the start of transcription in HIV-1, spanning the border of gpl20 and gp41). The RRE is necessary for Rev function; it contains a high affinity site for Rev; in all, approximately seven binding sites for Rev exist within the RRE RNA. Other lentiviruses (HIV-2, SIV, visna, CAEV) have similar RRE elements in similar locations within env, while HTLVs have an analogous RNA element (RXRE) serving the same purpose within their LTR; RRE is the binding site for Rev protein, while RXRE is the binding site for Rex protein. RRE (and RXRE) form complex secondary structures, necessary for specific protein binding.

[0823] Psi elements (PE) are a set of 4 stem-loop structures preceding and overlapping the Gag start codon which are the sites recognized by the cysteine histidine box, a conserved motif with the canonical sequence CysX2CysX4HisX4Cys (SEQ ID NO: 41), present in the Gag p7 MC protein. The Psi Elements are present in unspliced genomic transcripts but absent from spliced viral mRNAs.

[0824] SLIP, an ΓΤΤΤΤ slippery site, followed by a stem-loop structure, is responsible for regulating the - 1 ribosomal frameshift out of the Gag reading frame into the Pol reading frame. [0825] Cis-acting repressive sequences (CRS) are postulated to inhibit structural protein expression in the absence of Rev. One such site was mapped within the pol region of HIV- 1. The exact function has not been defined; splice sites have been postulated to act as CRS sequences.

[0826] Inhibitory/Instability RNA sequences (INS) are found within the structural genes of HIV- 1 and of other complex retroviruses. Multiple INS elements exist within the genome and can act independently; one of the best characterized elements spans nucleotides 414 to 631 in the gag region of HIV-1. The INS elements have been defined by functional assays as elements that inhibit expression posttranscriptionally. Mutation of the RNA elements was shown to lead to INS inactivation and up regulation of gene expression.

Genes and gene products

Essential for Replication

[0827] The genomic region (GAG) encoding the capsid proteins (group specific antigens). The precursor is the p55 myristylated protein, which is processed to p 17 (MAtrix), p24 (CApsid), p7 (NucleoCapsid), and p6 proteins, by the viral protease. Gag associates with the plasma membrane where the virus assembly takes place. The 55 kDa Gag precursor is called assemblin to indicate its role in viral assembly.

[0828] The genomic region, POL, encoding the viral enzymes protease, reverse transcriptase, RNAse, and integrase. These enzymes are produced as a Gag-Pol precursor polyprotein, which is processed by the viral protease; the Gag-Pol precursor is produced by ribosome frameshifting near the end of gag.

[0829] Viral glycoproteins (e.g., ENV) produced as a precursor (gpl60) which is processed to give a noncovalent complex of the external glycoprotein gpl20 and the transmembrane glyco protein gp41 . The mature gpl20-gp41 proteins are bound by non-covalent interactions and are associated as a trimer on the cell surface. A substantial amount of gpl20 can be found released in the medium. gpl20 contains the binding site for the CD4 receptor, and the seven transmembrane domain chemokine receptors that serve as co-receptors for HIV-1 .

[0830] The transactivator (TAT) of HIV gene expression is one of two essential viral regulatory factors (Tat and Rev) for HIV gene expression. Two forms are known, Tat-1 exon (minor form) of 72 amino acids and Tat-2 exon (major form) of 86 amino acids. Low levels of both proteins are found in persistently infected cells. Tat has been localized primarily in the nucleolus/nucleus by immunofluorescence. It acts by binding to the TAR RNA element and activating transcription initiation and elongation from the LTR promoter, preventing the LTR AATAAA polyadenylation signal from causing premature termination of transcription and polyadenylatio'n.

It is the first eukaryotic transcription factor known to interact with RNA rather than DNA and may have similarities with prokaryotic anti-termination factors. Extracellular Tat can be found and can be taken up by cells in culture.

[0831] The second necessary regulatory factor for HIV expression is REV. A 19 kDa phosphoprotein, localized primarily in the nucleolus/nucleus, Rev acts by binding to RRE and promoting the nuclear export, stabilization and utilization of the unspliced viral mRNAs containing RRE. Rev is considered the most functionally conserved regulatory protein of lentiviruses. Rev cycles rapidly between the nucleus and the cytoplasm.

Others

[0832] Viral infectivity factor (VIF) is a basic protein of typically 23 kDa. Promotes the infectivity but not the production of viral particles. In the absence of Vif the produced viral particles are defective, while the cell-to-cell transmission of virus is not affected significantly. Found in almost all lentiviruses, Vif is a cytoplasmic protein, existing in both a soluble cytosolic form and a membrane-associated form. The latter form of Vif is a peripheral membrane protein that is tightly associated with the cytoplasmic side of cellular membranes. In 2003, it was discovered that Vif prevents the action of the cellular APOBEC-3G protein which deaminates DNA:RNA heteroduplexes in the cytoplasm.

[0833] Viral Protein R (VPR) is a 96-amino acid (14 kDa) protein, which is incoiporated into the

virion. It interacts with the p6 Gag part of the Pr55 Gag precursor. Vpr detected in the cell is localized to the nucleus. Proposed functions for Vpr include the targeting the nuclear import of preintegration complexes, cell growth arrest, transactivation of cellular genes, and induction of cellular differentiation. In HIV-2, SIV-SMM, SIV- RCM, SIV-MND-2 and SIV-DRL the Vpx gene is apparently the result of a Vpr gene duplication event, possibly by recombination. [0834] Viral Protein (VPU)) is unique to HIV-1, SIVcpz (the closest SIV relative of HIV- 1), SIV-GSN, SIV-MUS, SIV- MON and SIV-DEN. There is no similar gene in HIV-2, SIV-SMM or other SIVs. Vpu is a 16 kDa (81 -amino acid) type I integral membrane protein with at least two different biological functions: (a) degradation of CD4 in the endoplasmic reticulum, and (b) enhancement of virion release from the plasma membrane of HIV-1-infected cells. Env and Vpu are expressed from a bicistronic mRNA. Vpu probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. It is phosphorylated by casein kinase II at positions Ser52 and Ser56. Vpu is involved in Env maturation and is not found in the virion. Vpu has been found to increase susceptibility of' HIV-1 infected cells to Fas killing.

[0835] NEF is amultifunctional 27-kDa myristylated protein produced by an ORF located at the 3 0 end of the primate lentiviruses. Other forms of Nef are known, including nonmyristylated variants. Nef is predominantly cytoplasmic and associated with the plasma membrane via the myristyl residue linked to the conserved second amino acid (Gly). Nef has also been identified in the nucleus and found associated with the cytoskeleton in some experiments. One of the first HIV proteins to be produced in infected cells, it is the most immunogenic of the accessory proteins. The nef genes of HIV and SIV are dispensable in vitro, but are essential for efficient viral spread and disease progression in vivo. Nef is necessary for the maintenance of high virus loads and for the development of AIDS in macaques, and viruses with defective Nef have been detected in some HIV-1 infected long term survivors. Nef downregulates CD4, the primary viral receptor, and MHC class I molecules, and these functions map to different parts of the protein. Nef interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. It increases viral infectivity. Nef contains PxxP motifs that bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of HIV but not for the downregulation of CD4.

[0836] VPX is a virion protein of 12 kDa found in HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2 and SIV-DRL and not in HIV-1 or other SIVs. This accessory gene is a homolog of HIV-1 vpr, and viruses with Vpx carry both vpr and vpx. Vpx function in relation to Vpr is not fully elucidated; both are incorporated into virions at levels comparable to Gag proteins through interactions with Gag p6. Vpx is necessary for efficient replication of SIV-SMM in PBMCs. Progression to AIDS and death in SIV-infected animals can occur in the absence of Vpr or Vpx. Double mutant virus lacking both vpr and vpx was attenuated, whereas the single mutants were not, suggesting a redundancy in the function of Vpr and Vpx related to virus pathogenicity.

Hepatitis A Viral Target Sequences 5' untranslated region contains IRES - internal ribosome entry site P I Region of genome - capsid proteins VP1 VP2 VP3 VP4

P2 Region of genome 2A 2B 2C

P3 Region of genome 3A 3B 3C - viral protease 3D - RNA polymerase

Hepatitis B Viral Target Sequences

[0837] Precursor Polypeptide encoding all HCV protein is produced and then spliced into functional proteins. The following are the proteins (coding regions) encoded:

C - core protein - coding region consists of a Pre-C and Core coding region X - function unclear but suspected to play a role in activation of viral transcription process P - RNA polymerase S - surface antigen - coding region consists of a Pre-S l, Pre-S2 and Surface antigen coding regions Hepatitis C Viral Target Sequences

[0838] Precursor Polypeptide encoding all HCV protein is produced and then spliced into functional proteins. The following are the proteins (coding regions) encoded:

RES - non-coding internal ribosome entry site (5' to polyprotein encoding sequence) 3' non-coding sequences - C region - encodes p22 a nucleocapsid protein El region - encodes gp35 envelope glycoprotein - important in cell entry E2 region - encodes gp70 envelope glycoprotein - important in cell entry NS 1 - encodes p7 - not necessary for replication but critical in viral morphogenesis NS2 - encodes p23 a transmembrane protein with protease activity NS3 - encodes p70 having both serine protease and RNA helicase activities NS4A - encodes p8 co-factor NS4B - encodes p27 cofactor - important in recruitment of other viral proteins NS5A - encodes p56/58 an interferon resistance protein - important in viral replication NS5B - encodes RNA polymerase

Herpes Simplex Virus Target Sequence

UL35 VP26 Γ681 Capsid protein LRP2 LRP2 Latency-related protein transcript

HPV Target Sequences LCR Viral long control region (location of early promoters) Keratinocyte/auxiliary enhancer P Promoter Early (E) gene promoter for subtype HPV16 P o Promoter Early (E) gene promoter for subtype HPV 18 P(,7o Promoter Late (L) gene promoter for HPV 16 P742 Promoter Late (L) gene promoter for HPV3 1

Influenza A Target Sequences

[0839] Influenza A is the most common flu virus that infects humans. The influenza A virion is made up of 8 different single stranded RNA segments which encodes 11-14 proteins. These segments can vary in sequence, with most variation occun ing in the hemagglutinin (H or HA) surface protein and neuraminidase (NA or N). The eight RNA segments (and the proteins they encode) are:

HA - encodes hemagglutinin (about'500 molecules of hemagglutinin are needed to make one virion).

A - encodes neuraminidase (about 100 molecules of neuraminidase are needed to make one virion).

NP encodes nucleoprotein.

M encodes two matrix proteins (the l and the M2) by using different reading frames from the same RNA segment (about 3000 matrix protein molecules are needed to make one virion). M42 is produced by alternative splicing, and can partially replace an M2.

NS encodes two distinct non-structural proteins (NS1 and NEP) by using different reading frames from the same RNA segment.

PA encodes an RNA polymerase; an alternate form is sometimes made through a ribosomal skip, with + 1 frameshift, reading through to the next stop codon. PB1 encodes an RNA polymerase, plus two other transcripts read from alternate start sites, named PB1-N40 and PB1-F2 protein (induces apoptosis) by using different reading frames from the same RNA segment.

PB2 encodes an RNA polymerase.

M. tuberculosis Target Sequences

[0840] The methods and composition described herein can be used to target M. tuberculosis and treat a subject suffering from an infection with M. tuberculosis.

Other

[0841] In some embodiments, the target gene is associated with multiple drug resistance (MDR), e.g., in bacterial infection. Infectious pathogens can use a number of mechanisms in attaining multi-drug resistance, e.g., no longer relying on a glycoprotein cell wall, enzymatic deactivation of antibiotics, decreased cell wall permeability to antibiotics, altered target sites of antibiotic, efflux pumps to remove antibiotics, increased mutation rate as a stress response, or a combination thereof.

IX. Targets: Gene Editing/Correction

[0842] Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, can be used to modulate genes (e.g., mutated genes) responsible for diseases. In some embodiments, the gene is modulated by editing or correcting a target gene, e.g., as described herein. In other embodiments, the human gene is modulated by delivery of one or more regulators/effectors (e.g., as described herein) inside cells to the target gene. For example, the genes described herein can be modulated, in vitro, ex vivo, or in vivo.

Table X- . Selected Diseases in which a gene can be therapeutically targeted.

o Kinases (cancer) o Energy metabolism (cancer) o CFTR (cystic fibrosis) o Color blindness o Hemochromatosis o Hemophilia o Phenylketonuria o Polycystic kidney disease o Sickle-cell disease o Tay-Sachs disease o Siderius X-linked mental retardation syndrome o Lysosomal storage disorders, e.g., Alpha-galactosidase A deficiency o Anderson-Fabry disease o Angiokeratoma Corporis Diffusum o CADASIL syndrome o Carboxylase Deficiency, Multiple, Late-Onset o Cerebelloretinal Angiomatosis, familial o Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy o Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy o Cerebroside Lipidosis syndrome o Choreoathetosis self-mutilation hyperuricemia syndrome o Classic Galactosemia o Crohn's disease, fibrostenosing o Phenylalanine Hydroxylase Deficiency disease, o Fabry disease o Hereditary coproporphyria o Incontinentia pigmenti o Microcephaly o Polycystic kidney disease o Rett's o Alpha- 1 antitrypsin deficiency o Wilson's Disease o Tyrosinemia o Frameshift related diseases o Cystic fibrosis o Triplet repeat diseases (also referred herein as trinucleotide repeat diseases)

[0843] Trinucleotide repeat diseases (also known as triplet repeat disease, trinucleotide repeat expansion disorders, triplet repeat expansion disorders, or codon reiteration disorders) are a set of genetic disorders caused by trinucleotide repeat expansion, e.g., a type of mutation where trinucleotide repeats in certain genes exceed the normal and/or stable threshold. The mutation can be a subset of unstable microsatellite repeats that occur in multiple or all genomic sequences. The mutation can increase the repeat count (e.g., result in extra or expanded repeats) and result in a defective gene, e.g., producing an abnormal protein. Trinucleotide repeats can be classified as insertion mutations or as a separate class of mutations. Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, can be used to modulate one or more genes (e.g., mutated genes) associated with a trinucleotide repeat disease, e.g., by reducing the number of (e.g., removing) the extra or expanded repeats, such that the normal or wild-type gene product (e.g., protein) can be produced.

[0844] Exemplary trinucleotide repeat diseases and target genes involved in trinucleotide repeat diseases are shown in Table IX-IA.

Table IX-IA. Exemplary trinucleotide repeat diseases and target genes involved in trinucleotide repeat diseases

[0845] Exemplary target genes include those genes involved in various diseases or conditions, e.g., cancer (e.g., kinases), energy metabolism, cystic fibrosis (e.g., CFTR), color blindness, hemochromatosis, hemophilia, phenylketonuria, polycystic kidney disease, Sickle-cell disease, Tay-Sachs disease, Siderius X-linked mental retardation syndrome, Lysosomal storage disorders (e.g., Alpha-galactosidase A deficiency), Anderson-Fabry disease, Angiokeratoma Corporis Diffusum, CADASIL syndrome, Carboxylase Deficiency, Multiple, Late-Onset, Cerebelloretinal Angiomatosis, familial, Cerebral aiteriopafhy with subcortical infarcts and leukoencephalopathy, Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Cerebroside Lipidosis syndrome, Choreoathetosis self-mutilation hyperuricemia syndrome, Classic Galactosemia, Crohn's disease, fibrostenosing, Phenylalanine Hydroxylase Deficiency disease, Fabry disease, Hereditary coproporphyria, Incontinentia pigmenti , . Microcephaly, Polycystic kidney disease, Rett's, Alpha- 1 antitrypsin deficiency, Wilson's Disease, Tyrosinemia, Frameshift related diseases, and Triplet repeat diseases.

[0846] Exemplary target genes and diseases are described in Tabic IX-2. The left hand column indentifies the disease and the right hand column identifies a gene for manipulation. (Table IX- 2 is provided in Anne IX-2).

[0847] Additional exemplary target genes include genes associated with diseases including, e.g., Crigler-Najjer syndrome, Glycogen storage disease type IV (GSD type IV), Familial hemophagocytic lymphohistiocytosis (FHL-Perforin deficiency), Ornithine transcarbamylase deficiency (OTC deficiency) or other Urea Cycle Disorders, Primary Hyperoxaluria, Leber congenital amaurosis (LCA), Batten disease, Chronic Granulomatous Disease, Wiskott-Aldrich syndrome, Usher Syndrome, and hemoglobinoapthies.

[0848] Crigler-Najjer syndrome. Crigler-Najjer syndrome is a severe condition characterized by high levels of bilirubin in the blood (hyperbilirubinemia). Bilirubin is produced when red blood cells are broken down. This substance is removed from the body only after it undergoes a chemical reaction in the liver, which converts the toxic form of bilirubin (unconjugated bilirubin) to a nontoxic form (conjugated bilirubin). People with Crigler-Najjar syndrome have a buildup of unconjugated bilirubin in their blood (unconjugated hyperbilirubinemia). Crigler-Najjar syndrome is divided into two types. Type 1 (CN1) is very severe and Type 2 (CN2) is less severe.

[0849] Mutations in the UGTl A 1gene can cause Crigler-Najjar syndrome. This gene provides instructions for making the bilirubin uridine diphosphate glucuronosyl transferase (bilirubin- UGT) enzyme, which is found primarily in liver cells and is necessary for the removal of bilirubin from the body. The bilirubin-UGT enzyme is involved in glucuronidation, in which the enzyme transfers glucuronic acid to unconjugated bilirubin, converting it to conjugated bilirubin. Glucuronidation makes bilirubin dissolvable in water so that it can be removed from the body.

[0850] Mutations in the UGT1 Al gene that cause Crigler-Najjar syndrome result in reduced or absent function of the bilirubin-UGT enzyme. People with C l have no enzyme function, while people with CN2 can have less than 20 percent of normal function. The loss of bilirubin-UGT function decreases glucuronidation of unconjugated bilirubin. This toxic substance then builds up in the body, causing unconjugated hyperbilirubinemia and jaundice.

[0851] Glycogen storage disease type IV. Glycogen storage disease type IV (also known as GSD type IV, Glycogenosis type IV, Glycogen Branching Enzyme Deficiency (GBED), polyglucosan body disease, or Amylopectinosis) is an inherited disorder caused by the buildup of a complex sugar called glycogen in the body's cells. The accumulated glycogen is structurally abnormal and impairs the function of certain organs and tissues, especially the liver and muscles.

[0852] Mutations in the GBE1 gene cause GSD IV. The GBE1 gene provides instructions for making the glycogen branching enzyme. This enzyme is involved in the production of glycogen, which is a major source of stored energy in the body. GBE1 gene mutations that cause GSD IV lead to a shortage (deficiency) of the glycogen branching enzyme. As a result, glycogen is not formed properly. Abnormal glycogen molecules called polyglucosan bodies accumulate in cells, leading to damage and cell death. Polyglucosan bodies accumulate in cells throughout the body, but liver cells and muscle cells are most severely affected in GSD IV. Glycogen accumulation in the liver leads to hepatomegaly and interferes with liver functioning. The inability of muscle cells to break down glycogen for energy leads to muscle weakness and wasting.

[0853] Generally, the severity of the disorder is linked to the amount of functional glycogen branching enzyme that is produced. Individuals with the fatal perinatal neuromuscular type tend to produce less than 5 percent of usable enzyme, while those with the childhood neuromuscular type may have around 20 percent of enzyme function. The other types of GSD IV are usually associated with between 5 and 20 percent of working enzyme. These estimates, however, vary among the different types.

[0854] Familial hemophagocytic lymphohistiocytosis. Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder in which the immune system produces too many activated immune cells (lymphocytes), e.g., T cells, natural killer cells, B cells, and macrophages (histiocytes). Excessive amounts of cytokines are also produced. This overactivation of the immune system causes fever and damages the liver and spleen, resulting in enlargement of these organs.

[0855] Familial hemophagocytic lymphohistiocytosis also destroys blood-producing cells in the bone marrow, a process called hemophagocytosis. The brain may also be affected in familial hemophagocytic lymphohistiocytosis. In addition to neurological problems, familial hemophagocytic lymphohistiocytosis can cause abnormalities of the heart, kidneys, and other organs and tissues. Affected individuals also have an increased risk of developing cancers of blood-forming cells (leukemia and lymphoma).

[0856] Familial hemophagocytic lymphohistiocytosis may be caused by mutations in any of several genes. These genes provide instructions for making proteins that help destroy or deactivate lymphocytes that are no longer needed. By controlling the number of activated lymphocytes, these genes help regulate immune system function.

[0857] Approximately 40 to 60 percent of cases of familial hemophagocytic lymphohistiocytosis are caused by mutations in the PRF1 or UNCI 3D genes. Smaller numbers of cases are caused by mutations in other known genes such as STX1 1 or STXBP2. The gene mutations that cause familial hemophagocytic lymphohistiocytosis can impair the body's ability to regulate the immune system. These changes result in the exaggerated immune response characteristic of this condition.

[0858] Ornithine transcarbamylase deficiency. Ornithine transcarbamylase deficiency (OTC) is an inherited disorder that causes ammonia to accumulate in the blood.

[0859] Mutations in the OTC gene cause ornithine transcarbamylase deficiency.

[0860] Ornithine transcarbamylase deficiency belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of reactions that occurs in liver cells. It processes excess nitrogen, generated when protein is used by the body, to make a compound called urea that is excreted by the kidneys. [0861] In ornithine transcarbamylase deficiency, the enzyme that starts a specific reaction within the urea cycle is damaged or missing. The urea cycle cannot proceed normally, and nitrogen accumulates in the bloodstream in the form of ammonia.

[0862] Ammonia is especially damaging to the nervous system, so ornithine transcarbamylase . deficiency causes neurological problems as well as eventual damage to the liver.

[0863] Other urea cycle disorders and associate genes include, e.g., N-Acetylglutamate synthase deficiency (NAGS), Carbamoyl phosphate synthetase I deficiency (CPS1), "AS deficiency" or citrullinemia (ASS), "AL deficiency" or argininosuccinic aciduria (ASL), and "Arginase deficiency" or argininemia (ARG).

[0864] Primary hyperoxaluria. Primary hyperoxaluria, e.g., primary hyperoxaluria type 1 (PH 1), is a rare, autosomal recessive inherited genetic condition in which an error in the glyoxylate metabolism pathway in the liver leads to an overproduction of oxalate, which crystalizes in soft tissues including the kidney, bone marrow, and eyes. The disease manifests as progressive deterioration of the kidneys, and treatment is a complicated double transplant of kidney (the damaged organ) and liver (the diseased organ).

[0865] Primary hyperoxaluria is caused by the deficiency of an enzyme that normally prevents the buildup of oxalate. There are two types of primary hyperoxaluria, distinguished by the enzyme that is deficient. People with type 1 primary hyperoxaluria have a shortage of a liver enzyme called alanine-glyoxylate aminotransferase (AGXT). Type 2 primary hyperoxaluria is characterized by a shortage of an enzyme called glyoxylate reductase/hydroxypyruvate reductase (GRHPR).

[0866] Mutations in the AGXT and GRHPR genes cause primary hyperoxaluria. The breakdown and processing of certain sugars and amino acids produces a glyoxylate. Normally, glyoxylate is converted to the amino acid glycine or to glycolate through the action of two enzymes, alanine-glyoxylate aminotransferase and glyoxylate reductase/hydroxypyruvate reductase, respectively. Mutations in the AGXT or GRHPR gene cause a shortage of these enzymes, which prevents the conversion of glyoxylate to glycine or glycolate. As levels of glyoxylate build up, it is converted to oxalate. Oxalate combines with calcium to form calcium oxalate deposits, which can damage the kidneys and other organs. [0867] In ah embodiment, the genetic defect in AGXT is corrected, e.g., by homologous recombination, using the Cas9 molecule and gRNA molecule described herein. For example, the functional enzyme encoded by the corrected AGXT gene can be redirected to its proper subcellular organelle. Though >50 mutations have been identified in the gene, the most common (40% in Caucasians) is a missense G170R mutation. This mutation causes the AGT enzyme to be localized to the mitochondria rather than to the peroxisome, where it must reside to perform its function. Other common mutations include, e.g., I244T (Canary Islands), F 521, G41R, G630A (Italy), and G588A (Italy).

[0868] In an embodiment, one or more genes encoding enzymes upstream in the glyoxylate metabolism pathway are targeted, using the Cas9 molecule and gRNA molecule described herein. Exemplary targets include, e.g., glycolate oxidase (gene HAOl, OMIM ID 605023). Glycolate oxidase converts glycolate into glyoxylate, the substrate for AGT. Glycolate oxidase is only expressed in the liver and, because of its peroxisomal localization, makes it a suitable target in this metabolic pathway. In an embodiment, a double-strand break in the HAO gene is introduced and upon repair by NHEJ a frame-shift results in a truncated protein. In an embodiment, a transcriptional repressor (e.g., a transcriptional repressor described herein) is delivered as a payload to the HAOl gene to reduce the expression of HAOl.

[0869] lAiber congenital amaurosis. Leber congenital amaurosis (LCA) is an eye disorder that primarily affects the retina. People with this disorder typically have severe visual impairment beginning in infancy. The visual impairment tends to be stable, although it may worsen very slowly over time. At least 13 types of Leber congenital amaurosis have been described. The types are distinguished by their genetic cause, patterns of vision loss, and related eye abnormalities.

[0870] Leber congenital amaurosis can result from mutations in at least 14 genes, all of which are necessary for normal vision. These genes play a variety of roles in the development and function of the retina. For example, some of the genes associated with this disorder are necessary for the normal development of photoreceptors. Other genes are involved in phototransduction. Still other genes play a role in the function of cilia, which are necessary for the perception of several types of sensory input, including vision. [0871] Mutations in any of the genes associated with Leber congenital amaurosis (e.g., A1PL1, CEP290, CRB 1, CRX, GUCY2D, IMPDH1, LCA5, LRAT, RD3, RDH12, RPE65, RPGRIP1, SPATA7, TULP1) can disrupt the development and function of the retina, resulting in early vision loss. Mutations in the CEP290, CRB1, GUCY2D, and RPE65 genes are the most common causes of the disorder, while mutations in the other genes generally account for a smaller percentage of cases.

[0872] Batten disease. Batten disease or juvenile Batten disease is an inherited disorder that primarily affects the nervous system. After a few years of normal development, children with this condition develop progressive vision loss, intellectual and motor disability, and seizures.

[0873] Juvenile Batten disease is one of a group of disorders known as neuronal ceroid lipofuscinoses (NCLs). These disorders all affect the nervous system and typically cause progressive problems with vision, movement, and thinking ability. Some people refer to the entire group of NCLs as Batten disease, while others limit that designation to the juvenile form of the disorder. The different types of NCLs are distinguished by the age at which signs and symptoms first appear.

[0874] Most cases of juvenile Batten disease are caused by mutations in the CLN3 gene. These mutations can disrupt the function of cellular structures called lysosomes. Lysosome malfunction leads to a buildup of lipopigments within these cell structures. These accumulations occur in cells throughout the body, but neurons in the brain seem to be particularly vulnerable to the damage caused by lipopigments. The progressive death of cells, especially in the brain, leads to vision loss, seizures, and intellectual decline in people with juvenile Batten disease.

[0875] A small percentage of cases of juvenile Batten disease are caused by mutations in other genes (e.g., ATP13A2, CLN5, PPT1, TPP1). Many of these genes are involved in lysosomal function, and when mutated, can cause this or other forms of NCL.

[0876] Chronic granulomatous disease. Chronic granulomatous disease is a disorder that causes the immune system to malfunction, resulting in a form of immunodeficiency. Individuals with chronic granulomatous disease have recurrent bacterial and fungal infections. People with this condition often have areas of inflammation (granulomas) in various tissues that can be damaging to those tissues. e features of chronic granulomatous disease usually first appear in childhood, although some individuals do not show symptoms until later in life. [0877] Mutations in the CYBA, CYBB, NCF1, NCF2, or NCF4 gene can cause chronic granulomatous disease. There are five types of this condition that are distinguished by the gene that is involved. The proteins produced from the affected genes are subunits of NADPH oxidase, which plays an important role in the immune system. Specifically, NADPH oxidase is primarily active in phagocytes. Within phagocytes, NADPH oxidase is involved in the production of superoxide, which plays a role in .killing foreign invaders and preventing them from reproducing in the body and causing illness. NADPH oxidase also regulates the activity of neutrophils, which play a role in adjusting the inflammatory response to optimize healing and reduce injury to the body.

[0878] Mutations in the CYBA, CYBB, NCF1, NCF2, and NCF4 genes result in the production of proteins with little or no function or the production of no protein at all. Without any one of its subunit proteins, NADPH oxidase cannot assemble or function properly. As a result, phagocytes are unable to kill foreign invaders and neutrophil activity is not regulated. A lack of NADPH oxidase leaves affected individuals vulnerable to many types of infection and excessive inflammation.

[0879] Wiskott-Aldrich syndrome. Wiskott-Aldrich syndrome is characterized by abnormal immune system function (immune deficiency) and a reduced ability to form blood clots. This condition primarily affects males. Individuals with Wiskott-Aldrich syndrome have microthrombocytopenia, which is a decrease in the number and size of blood cells involved in clotting (platelets), which can lead to easy bruising or episodes of prolonged bleeding following minor trauma. Wiskott-Aldrich syndrome causes many types of white blood cells to be abnormal or nonfunctional, leading to an increased risk of several immune and inflammatory disorders. Many people with this condition develop eczema, an inflammatory skin disorder characterized by abnormal patches of red, irritated skin. Affected individuals also have an increased susceptibility to infection. People with Wiskott-Aldrich syndrome are at greater risk of developing autoimmune disorders. The chance of developing some types of cancer, such as cancer of the immune system cells (lymphoma), is also greater in people with Wiskott-Aldrich syndrome.

[0880] Mutations in the WAS gene cause Wiskott-Aldrich syndrome. The WAS gene provides instructions for making WASP protein, which is found in all blood cells. WASP is involved in relaying signals from the surface of blood cells to the actin cytoskeleton. WASP signaling activates the cell when it is needed and triggers its movement and attachment to other cells and tissues (adhesion). In white blood cells, this signaling allows the actin cytoskeleton to establish the interaction between cells and the foreign invaders that they target (immune synapse).

[0881] WAS gene mutations that cause Wiskott-Aldrich syndrome lead to a lack of any functional WASP. Loss of WASP signaling disrupts the function of the actin cytoskeleton in developing blood cells. White blood cells that lack WASP have a decreased ability to respond to their environment and form immune synapses. As a result, white blood cells are less able to respond to foreign invaders, causing many of the immune problems related to Wiskott-Aldrich syndrome. Similarly, a lack of functional WASP in platelets impairs their development, leading to reduced size and early cell death.

[0882] Usher syndrome. Usher syndrome is a condition characterized by hearing loss or deafness and progressive vision loss. The loss of vision is caused by retinitis pigmentosa (RP), which affects the layer of light-sensitive tissue at the back of the eye (the retina). Vision loss occurs as the light-sensing cells of the retina gradually deteriorate.

[0883] Three major types of Usher syndrome, designated as types I (subtypes IA through IG), II (subtypes IIA, I , and IIC), and III, have been identified. These types are distinguished by their severity and the age when signs and symptoms appear.

[0884] Mutations in the CDH23, CLRN , GPR98, MY07A, PCDH15, USH1C, USH1G, and USH2A genes can cause Usher syndrome. The genes related to Usher syndrome provide instructions for making proteins that play important roles in normal hearing, balance, and vision. They function in the development and maintenance of hair cells, which are sensory cells in the inner ear that help transmit sound and motion signals to the brain. In the retina, these genes are also involved in determining the structure and function of light-sensing cells called rods and cones. In some cases, the exact role of these genes in hearing and vision is unknown. Most of the mutations responsible for Usher syndrome lead to a loss of hair cells in the inner ear and a gradual loss of rods and cones in the retina. Degeneration of these sensory cells causes hearing loss, balance problems, and vision loss characteristic of this condition.

[0885] Usher syndrome type I can result from mutations in the CDH23, MY07A, PCDH15, USH1C, or USH1G gene. Usher syndrome type II can be caused by mutations in, e.g., USH2A or GPR98 (also called VLGR1) gene. Usher syndrome type III can be caused by mutations in e.g., CLR 1.

[0886] Hemoglobinopathies. Hemoglobinopathies are a group of genetic defects that result in abnormal structure of one of the globin chains of the hemoglobin molecule. Exemplary hemoglobinopathies include, e.g., sickle cell disease, alpha thalassemia, and beta thalassemia.

[0887] In an embodiment, a genetic defect in alpha globulin or beta globulin is corrected, e.g., by homologous recombination, using the Cas9 molecule and gRNA molecule described herein.

[0888] n an embodiment, a hemoglobinopathies-associated gene is targeted, using the Cas9 molecule and gRNA molecule described herein. Exemplary targets include, e.g., genes associated with control of the gamma-globin genes. In an embodiment, the target is BCL1 1A.

[0889] Fetal hemoglobin (also hemoglobin F or HbF or α2γ 2) is a tetramer of two adult alpha- globin polypeptides and two fetal beta-like gamma-globin polypeptides. HbF is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old. Functionally, fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream.

[0890] In newborns, fetal hemoglobin is nearly completely replaced by adult hemoglobin by approximately 6 months postnatally. In adults, fetal hemoglobin production can be reactivated pharmacologically, which is useful in the treatment of diseases such as hemoglobinopathies. For example, in certain patients with hemoglobinopathies, higher levels of gamma-globin expression can partially compensate for defective or impaired beta-globin gene production, which can ameliorate the clinical severity in these diseases. Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of hemoglobinopathies, e.g., beta- thalassemia major and sickle cell anemia.

[0891] Increased HbF levels or F-cell can be associated reduced BCL1 1A expression in cells. The BCL1 1A gene encodes a multi-zinc finger transcription factor. In an embodiment, the expression of BCL1 1A is modulated, e.g., down-regulated. In an embodiment, the BCL1 1A gene is edited. In an embodiment, the cell is a hemopoietic stem cell or progenitor cell. Sickle cell diseases

[0892] Sickle cell disease is a group of disorders that affects hemoglobin. People with this disorder have atypical hemoglobin molecules (hemoglobin S), which can distort red blood cells into a sickle, or crescent, shape. Characteristic features of this disorder include a low number of red blood cells (anemia), repeated infections, and periodic episodes of pain.

[0893] Mutations in the HBB gene cause sickle cell disease. T e HBB gene provides instructions for making beta-globin. Various versions of beta-globin result from different mutations in the HBB gene. One particular HBB gene mutation produces an abnormal version of beta-globin known as hemoglobin S (HbS). Other mutations in the HBB gene lead to additional abnormal versions of beta-globin such as hemoglobin C (HbC) and hemoglobin E (HbE). HBB gene mutations can also result in an unusually low level of beta-globin, i.e., beta thalassemia.

[0894] In people with sickle cell disease, at least one of the beta-globin subunits in hemoglobin is replaced with hemoglobin S . In sickle cell anemia, which is a common form of sickle cell disease, hemoglobin S replaces both beta-globin subunits in hemoglobin. In other types of sickle cell disease, just one beta-globin subunit in hemoglobin is replaced with hemoglobin S. The other beta-globin subunit is replaced with a different abnormal variant, such as hemoglobin C. For example, people with sickle-hemoglobin C (HbSC) disease have hemoglobin molecules with hemoglobin S and hemoglobin C instead of beta-globin. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S-beta thalassemia (HbSBetaThal) disease.

Alpha thalassemia

[0895] Alpha thalassemia is a blood disorder that reduces the production of hemoglobin. In people with the characteristic features of alpha thalassemia, a reduction in the amount of hemoglobin prevents enough oxygen from reaching the body's tissues. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue, and more serious complications.

[0896] Two types of alpha thalassemia can cause health problems. The more severe type is hemoglobin Bart hydrops fetalis syndrome or Hb Bart syndrome. The milder form is HbH disease. Hb Bart syndrome is characterized, e.g., by hydrops fetalis, a condition in which excess fluid builds up in the body before birth. HbH disease can cause, e.g., mild to moderate anemia, hepatosplenomegaly, and yellowing of the eyes and skin (jaundice).

[0897] Alpha thalassemia typically results from deletions involving the HBA1 and HBA2 genes. Both of these genes provide instructions for making alpha-globin, which is a subunit of hemoglobin. The different types of alpha thalassemia result from the loss of some or all of these . alleles.

[0898] Hb Bart syndrome can result from the loss of all four alpha-globin alleles. HbH disease can be caused by a loss of three of the four alpha-globin alleles. In these two conditions, a shortage of alpha-globin prevents cells from making normal hemoglobin. Instead, cells produce abnormal forms of hemoglobin, i.e., hemoglobin Bart (Hb Bart) or hemoglobin H (HbH), which cannot effectively carry oxygen to the body's tissues. The substitution of Hb Bart or HbH for normal hemoglobin can cause anemia and the other serious health problems associated with alpha thalassemia.

[0899] Two additional variants of alpha thalassemia are related to a reduced amount of alpha- globin. A loss of two of the four alpha-globin alleles can result in alpha thalassemia trait. People with alpha thalassemia trait may have unusually small, pale red blood cells and mild anemia. A loss of one alpha-globin allele can be found in alpha thalassemia silent carriers.

Beta thalassemia

[0900] Beta thalassemia is a blood disorder that reduces the production of hemoglobin. In people with beta thalassemia, low levels of hemoglobin lead to a lack of oxygen in many parts of the body. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue, and more serious complications. People with beta thalassemia are at an increased risk of developing abnormal blood clots.

[0901] Beta thalassemia is classified into two types depending on the severity of symptoms: thalassemia major (also known as Cooley's anemia) and thalassemia intermedia. Of the two types, thalassemia major is more severe.

[0902] Mutations in the HBB gene cause beta thalassemia. The HBB gene provides instructions for making beta-globin. Some mutations in the HBB gene prevent the production of any beta- globin. The absence of beta-globin is referred to as beta-zero (B°) thalassemia. Other HBB gene mutations allow some beta-globin to be produced but in reduced amounts, i.e., beta-plus (B+) thalassemia. People with both types have been diagnosed with thalassemia major and thalassemia intermedia.

[0903] In an embodiment, a Cas9 molecule/gRNA molecule complex targeting a first gene is used to treat a disorder characterized by second gene, e.g., a mutation in a second gene. By way of example, targeting of the first gene, e.g., by editing or payload delivery, can compensate for, or inhibit further damage from, the affect of a second gene, e.g., a mutant second gene. In an embodiment the allele(s) of the first gene carried by the subject is not causative of the disorder.

Table IX-3. Selected Disorders and Targets for Compensatory Targeting Comment Miiti-genetic origin. Factor H aHUS due to fH deficiency. deficiency is a risk factor. C5 antibody has been shown Controlling the complement to vastly improve prognosis. cascade, through fi l Can approach disease directly upregulation or C5 through increasing fH levels downregulation, may have a or controlling complement beneficial effect. through C5 downregulation.

Indication Devices: Graft orthopedics- Parkinson's Allergic Epilepsy Barrett's stent, hcaling/wou articular Disease rhinitis esophagus, pacemaker, nd cartilage Stomach hernia mesh- healing/prev repair, ulcer, gastritis local delivery ention of arthritis to prevent fibrosis restenosis/ fibrosis Target mTORC2, VEGF IL- SNCA, HI H I receptors H2 receptor others LRRK2, Receptors CNS pylorus, EIF4GI nasal esophagus mucosa Upregulate down- up-regiilate tip-regulate up-regulate down- up-regulate down-regulate / regulate or fix regulate ownr ul mutations ate Level of everolimus VEGF local animal model H I-anti¬ animal H2-specific evidence: administratio of cartilage histamines, models antihistamines. Market n aids in repair e.g. Zyrtec e g - proxy or tracheal omeprazole. animal transplant etc. model animal models Comment Embodiments Useful, e.g.. In include, e.g.. in the embodiments. embodiments, local delivery promoting the subject the subject is to tissue via wound sufferes from treated for device or healing arthritis or is late-stage injection to (burns, etc); in need of barren's. prevent Embodiments healing after fibrosis, include, e.g., injury. In restenosis local delivery embodiments, of growth chondrocytes factors are targeted post-injury to promote healing.

[0904] In an embodiment, Cas9 molecules, gRNA molecules, and/or Cas9 molecule/gRNA molecule complexes can be used to activate genes that regulate growth factors, such as up regulation of Epo to drive RBC production.

[0905] In an embodiment, Cas9 molecules, gRNA molecules, and/or Cas9 molecule/gRNA molecule complexes can be used to target, e.g., result in repression of, knockout of, or alteration of promoter for key transcription factors, such as BCLl 1A and KLFl for up-regulating of fetal hemoglobin, e.g., for cure for sickle cell anemia and thalassemia.

[0906] Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, as described herein, can be used to edit/correct a target gene or to deliver a regulator/effector inside cells, e.g., as described herein, at various subcellular locations. In some embodiments, the location is in the nucleus. In some embodiments, the location is in a sub-nuclear domain, e.g., the chromosome territories, nucleolus, nuclear speckles, Cajal bodies, Gems (gemini of Cajal bodies), or promyelocytic leukemia (PML) nuclear bodies. In other embodiments, the location is in the mitochondrion.

[0907] Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, as described herein, can be used to edit/correct a target gene or to deliver a regulator/effector inside cells, as described herein, at various time points

[0908] For example, the editing/correction or delivery can occur at different phases of cell cycle, e.g., G O phase, Interphase (e.g., G l phase, S phase, G2 phase), or M phase. As another example, the editing/correction or delivery can occur at different stages of disease progression, e.g., at latent stage or active stage of a disorder (e.g., viral infection), or at any stage or subclassification of a disorder (e.g., cancer).

[0909] Methods of the invention allow for the treatment of a disorder characterized by unwanted cell proliferation, e.g., cancer. In an embodiment, cancer cells are manipulated to make them more susceptible to treatment or to endogenous immune surveillance. In an embodiment a cancer cell is modulated to make it more susceptible to a therapeutic. In an embodiment, a cancer cell is manipulated so as to increase the expression of a gene that increases the ability of the immune system to recognize or kill the cancer cell. E.g., a Cas9 molecule/gRNA molecule complex can be used to deliver a payload, or edit a target nucleic acid so as to increase the expression of an antigen, e.g., in the case where the cancer cell has downregulated expression of the antigen. In an embodiment, a payload, e.g., a payload comprising a transcription factor or other activator of expression is delivered to the cancer cell. In an embodiment, an increase in expression is effected by cleavage of the target nucleic acid, e.g., cleavage and correction or alteration of the target nucleic acid by a template nucleic acid. In an embodiment, a payload that overrides epigenetic silencing, e.g., a modulator of methylation, is delivered.

[0910] In an embodiment, the treatment further comprises administering a second anti-cancer , therapy, e.g., immunotherapy, e.g., an antibody that binds the upregulated antigen.

[0911] In an embodiment, methods described herein, e.g., targeting of a genomic signature, e.g., a somatic translocation, can be used to target the Cas9 molecule/gRNA molecule to a cancer cell.

[0912] In another aspect, the invention features a method of immunizing a subject against an antigen. The method comprises using a method described herein to promote the expression of the antigen from a cell, e.g., a blood cell, such that the antigen promotes an immune response. In an embodiment, the cell is manipulated ex vivo and then returned or introduced into the subject.

X. Modified Nucleosides, Nucleotides, and Nucleic Acids

[0913] Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRN A, but also other forms of RNA, e.g., mRNA, RNAi, or siRN A. As described herein "nucleoside" is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, "nucleotide" is defined as a nucleoside further comprising a phosphate group.

[0914] Modified nucleosides and nucleotides can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar; (iii) wholesale replacement of the phosphate moiety with "dephospho" linkers; (iv) modification or replacement of a naturally occurring nucleobase; (v) replacement or modification of the ribose-phosphate backbone; (vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and (vii) modification of the sugar.

[0915] The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an embodiment, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule are replaced with phosphorothioate groups.

[0916] In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a "modified nucleic acid." In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides. [0917] Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.

[0918] In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.

Definitions of Chemical Groups

[09 ] As used herein, "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

[0920] As used herein, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

[0921] As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. [0922] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.

[0923] As used herein, "arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

[0924] As used herein, "cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non- aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.

[0925] As used herein, "heterocyclyl" refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.

[0926] As used herein, "heteroaryl" refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

Phosphate Backbone Modifications

The Phosphate Group

[0927] In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. [0928] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the S" configuration (herein Sp).

[0929] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).

[0930] The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). T e replacement can occur at either linking oxygen or at both of the linking oxygens.

Replacement of the Phosphate Group

[0931] The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.

[0932] Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

Replacement of the Ribophosphate Backbone

[0933] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

[0934] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.

[0935] Examples of "oxy"-2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar);

polyethyleneglycols (PEG), 0(CH 2CH20 ) CH2CH 2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2' hydroxyl group modification can include "locked" nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a Ci- alkylene or C - heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH 2) -amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2' hydroxyl group modification can include the methoxyethyl group (MOE),

(OCH2CH2OCH , e.g., a PEG derivative).

[0936] "Deoxy" modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino

(wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH H) CH2CH - amino (wherein amino can be, e.g., as described herein), -NHC(0)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

[0937] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the Γ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C- . These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.

[0938] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3'- 2')).

Modifications on the Nucleobase

[0939] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

Uracil

[0940] n some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio- uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- u,ridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3- methyl-uridine (m U), 5-methoxy-uridine (mo U), uridine 5-oxyacetic acid (cmo5U), uridine 5- oxyacetic acid methyl ester (mcmo^U), 5-carboxymethyl-uridine (cm U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5U), 5-methoxycarbonylmethyl-uridine (mcm U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- methylaminomethyl-uridine (mnm U), 5-methylaminomethyl-2-thio-uridine (mnm s2U), 5- methylaminomethyl-2-seleno-uridine (mnm se U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm 5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm \s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (xcm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Trn 5s2U), l-taurinomethyl-4-

thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-

methyl-pseudouridine (ι 'ψ) 5-methyl-2-thio-uridine (m s2U), l-methyl-4-thio-pseudouridine (m's | ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (mV ), 2-thio-l-methyl- pseudouridine, 1-methyl- -deaza-pseudouridine, 2-thio- 1-methyl- 1-deaza-pseudouridine, dihydroundine (D), dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio- uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp U), l-methyl-3-(3-amino-3- carboxypropyOpseudouridine 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethy])-2-thio-uridine (inm s2U), a-thio-uridine, 2'-0-methyl-uridine (Urn),

5,2'-0-dimethyl-uridine (m Um), 2'-0-methyl-pseudouridine (ψ ), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mcm 5Um), 5-carbamoylmethyl-2'-0- methyl-uridine (ncm Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm Um), 3,2'-0-dimethyl-uridine (m Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (in 5Um), l-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

Cytosine

[0941] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m C), N4-acetyl-cytidine (act), 5- formyl-cytidine (f C), N4-methyl-cytidine (m C), 5-methyl-cytidine (m C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine (k C), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine ( Cm), N4-acetyl-2'-0- methyl-cytidine (ac Cm), N4,2'-0-dimethyl-cytidine (m Cm), 5-formyl-2'-0-methyl-cytidine (f 4 Cm), N4,N4,2'-0-trimethyl-cytidine (m 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine. Adenine

[0942] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino- purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloiO-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza- 8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m'A), 2-methyl-adenine (m A), N6-methyl-adenosine (m A), 2-methylthio-N6-methyl-adenosine (ms2m A), N6- isopentenyl-adenosine (i A), 2-methylthio-N6-isopentenyl-adenosine (ms i A), N6-(cis- hydroxyisopentenyl)adenos 'ine (io A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io A), N6-glycinylcarbamoyl-adenosine (g A), N6-threonylcarbamoyl-adenosine (t A), N6- methyl-N6-threonylcarbamoyl-adenosine (m t A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms g A), N6,N6-dimethyl-adenosine (m A), N6-hydroxynorvalylcarbamoyl- adenosine (hn A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn A), N6- acetyl-adenosine (ac A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2'-0-methyl-adenosine (Am), N ,2'-0-dimethyl-adenosine (m Am), N -Methyl-2'- deoxyadenosine, N6,N6,2'-0-trimethyl-adenosine (m Am), l,2'-0-dimethyl-adenosine (m'Am), 2'-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8- azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

Guanine

[0943] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1- methyl-inosine (m'l), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyo sine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undemriodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- + deaza-guanosine (preQ0), 7-aminomethyI-7-deaza-guanosine (preQi), archaeosine (G ), 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m'G), N2-methyl-guanosine (m G), N2,N2-dimethyl-guanosine (m 2G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m ,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2- dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methyl-2'-0- methyl-guanosine (m¾m), N2,N2-dimethyl-2'-0-methyl-guanosine (m Gm), l-methyl-2'-0- methyl-guanosine (m'Gm), N2,7-dimethyl-2'-0-methyl-guanosine (m ,7Gm), 2'-0-methyl- inosine (Im), l,2'-0-dimethyl-inosine (m'lm), 0 6-phenyl-2'-deoxyinosine, 2'-0-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, 0 -methy]-guanosine, 0 -Methyl-2'-deoxyguanosine, 2'- F-ara-guanosine, and 2'-F-guanosine.

Modified gRNAs

[0944] n some embodiments, the modified nucleic acids can be modified gRNAs. n some embodiments, gRNAs can be modified at the 3' end. In this embodiment, the gRNAs can be modified at the 3' terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as sown below:

wherein "U" can be an unmodified or modified uridine.

[0945] In another embodiment, the 3' terminal U can be modified with a 2' 3' cyclic phosphate as shown below:

wherein "U" can be an unmodified or modified uridine. [0946] In some embodiments, the gRNA molecules may contain 3' nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7- deaza-adenosine, can be incoiporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2' OH- group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2'-sugar modified, such as, 2-F 2'-0-methyl, thymidine (T), 2'-0- methoxyethyl-5-methyluridine (Teo), 2'-0-methoxyethyladenosine (Aeo ), 2'-0-methoxyethyl- 5-methylcytidine (m5Ceo ), and any combinations thereof.

[0947] n an embodiment, a one or more or all of the nucleotides in single stranded overhang of an RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.

XI. Linkers

[0948] In some embodiments, the payload can be linked to the Cas9 molecules or the gRNA, e.g., by a covalent linker. This linker may be cleavable or non-cleavable. In some embodiments, a cleavable linker may be used to release the payload after transport to the desired target.

[0949] Linkers can comprise a direct bond or an atom such as, e.g., an oxygen (O) or sulfur (S), a unit such as -NR- wherein R is hydrogen or alkyl, -C(0)-, -C(0)0-, -C(0)NH-, SO, S0 2, - SO N - or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or . unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, heteroarylalkyl. In some embodiments, one or more methylenes in the chain of atoms can be replaced with one or more of

O, S, S(O), S0 2, -S0 NH-, -NR-, -C(O)-, -C(0)0-, -C(0)NH-, a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic.

Non-cleavable linkages

[0950] n some embodiments, the payload is attached to the Cas9 molecule or gRNA through a linker that is itself is stable under physiological conditions, such as an alkylene chain, and does not result in release of the payload from the Cas9 molecule and/or gRNA for at least 2, 3, 4, 5,

10, 15, 24 or 48 hours or for at least 1, 2, 3, 4, 5,or 10 days when administered to a subject. In some embodiments, the payload and the Cas9 molecule and/or gRNA comprise residues of a functional groups through which reaction and linkage of the payload to the Cas9 molecule or gRNA was achieved. In some embodiments, the functional groups, which may be the same or different, terminal or internal, of the payload or Cas9molecule and/or gRNA comprise an amino, acid, imidazole, hydroxyl, thio, acyl halide, -HC=CH-, ≡C group, or derivative thereof. In some embodiments, the linker comprises a hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or -0-, -C(=X)-

(wherein X is NR,, O or S), -NR , -NR,C(0)-, -C(0)NRi-, -S(0)„-, -NRiS(0) -,- S(0) nNR, -, - NR C(0 )-NR -; and R , independently for each occurrence, represents H or a lower alkyl and wherein n is 0, 1, or 2.

[0951] In some embodiments, the linker comprises an alkylene moiety or a heteroalkylene moiety (e.g., an alkylene glycol moiety such as ethylene glycol). In some embodiments, a linker comprises a poly-L-glutamic acid, polylactic acid, poly(ethyleneimine), an oligosaccharide, an amino acid (e.g., glycine), an amino acid chain, or any other suitable linkage. The linker groups can be biologically inactive, such as a PEG, polyglycolic acid, or polylactic acid chain. In certain embodiments, the linker group represents a derivatized or non-derivatized amino acid (e.g., glycine). Cleavable Linkages

[0952] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In one embodiment, the cleavable linking group is cleaved at least 10 times or more, or at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0953] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0954] A cleavable linkage group, such as a disulfide bond (-S-S-) can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7 . 1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH. A linker can include a cleavable linking group that is cleavable by a particular enzyme.

[0955] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. The candidate cleavable linking group can also be tested for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals.

[0956] In some embodiments, the cleavable linkers include redox cleavable linkers, such as a disulfide group (-S-S-) and phosphate cleavable linkers, such as, e.g., -0-P(0)(OR)-0-, -O- P(S)(OR)-0-, -0-P(S)(SR)-0-, -S-P(0)(OR)-0-, -0-P(0)(OR)-S-, -S-P(0)(OR)-S-, -O- P(S)(OR)-S-, -S-P(S)(OR)-0-, -0-P(0)(R)-0-, -0-P(S)(R)-0-, -S-P(0)(R)-0-, -S-P(S)(R)-0-, - S-P(0)(R)-S-, -OP(S)(R)-S-, wherein R is hydrogen or alkyl.

Acid Cleavable Linking Groups

[0957] Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In some embodiments, acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C(=N)N-, -C(0)0-, or -OC(O)-.

Ester-Based Linking Groups

[0958] Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula -C(0)0-, or -OC(O)-.

X I. Formulations and Delivery

[0959] Exemplary formulations and methods for delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component are described herein, e.g., in Table XII- 1. .

Table X -1: DELIVERY SUMMARY Vector into Non- Integration Molecule Dividing Expression Delivered Cells Physical YES Transient NO Nucleic Acids and Proteins Viral Retrovirus NO Stable YES RNA

Lentivirus YES Stable YES/NO RNA with modifications Adenovirus YES Transient NO DNA

Adeno- YES Stable NO DNA Associated Virus (AAV) Vaccinia Virus YES Very NO DNA Transient Herpes YES Stable NO DNA Simplex Virus Depends on Nucleic Non- Viral Cationic . YES Transient Liposomes what is Acids delivered Proteins Polymeric YES Transient Depends on Nucleic Nanoparticles what is Acids delivered Proteins BIOLOGICAL Attenuated YES Transient NO Nucleic NON-VIRAL Bacteria Acids DELIVERY VEHICLES Engineered YES Transient NO Nucleic Bacteriophages Acids

Mammalian YES Transient NO Nucleic Virus-like Acids Particles Biological YES Transient NO Nucleic liposomes: Acids Erythrocyte Ghosts and Exosomes DELIVERY VEHICLES

[0960] In an embodiment, the delivery vehicle is a physical vehicle. In an embodiment, the vehicle is low density ultrasound. For example, microbubbles containing payload (e.g., made of biocompatible material such protein, surfactant, or biocompatible polymer or lipid shell) can be used and the microbubbles can be destructed by a focused ultrasound bean during microvascular transit. In sembodiments, the vehicle is electroporation. For example, naked nucleic acids or proteins can be delivered by electroporation, e.g., into cell suspensions or tissue environment, such as retina and embryonic tissue. In an embodiment, the vehicle is needle or jet injection. For example, naked nucleic acids or protein can be- injected into, e.g., muscular, liver, skin, brain or heart tissue.

[0961] In an embodiment, the delivery vehicle is a viral vector. Types of viruses include, e.g., retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, and herpes simplex viruses.

[0962] In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vectors can be pseudotyped with different/alternative viral envelope glycoproteins; engineered with cell type-specific receptors (e.g., genetically modification of viral envelope glycoproteins to incorporate targeting ligands such as peptide ligands, single chain antibodies, growth factors); and/or engineered to have a molecular bridge with dual specificities with one end recognizing viral glycoproteins and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibodies, avidin-biotin and chemical conjugation).

[0963] In an embodiment, the viral vector achieves cell type specific expression. For example, tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cells. The specificity of the vectors can also be mediated by microRNA-dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of viral vector and target cell membrane. For example, fusion proteins such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, certain viruses that require the breakdown of the cell wall (during cell division) will not infect non-diving cell. Incorporated nuclear localization peptides into the matrix proteins of the virus allow transduction into non-proliferating cells.

[0964] In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non- viral vector is an inorganic nanoparticle (e.g., attached to the payload to the surface of the nanoparticle). Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g.,

Fe Mn0 2), silica (e.g., can integrate multi-functionality, e.g., conjugate the outer surface of the nanoparticle with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload and internal magnetic component, mesaporous silica nanoparticles with a positive charged polymer loaded with chloioquine to enhance transfection of the non-viral vector in vitro, high density lipoproteins and gold nanoparticles, gold nanoparticles coated with payload which gets released when nanoparticles are exposed to increased temperature by exposure to near infrared light, gold, iron or silver nanoparticles with surface modified with polylysine or another charge polymer to capture the nucleic acid cargo. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.

[0965] Exemplary lipids and polymers for gene transfer are shown below in Tables XII-2 and XII-3.

[0966] Exemplary lipids for gene transfer are shown below in Table XII-2.

Table XII-2: Lipids Used for Gene Transfer [0967] Exemplary polymers for gene transfer are shown below in Table XII-3.

Table XII-3: Polymers Used for Gene Transfer Poly(amidoethylenimine) SS-PAEI Triethylenetetramine TETA Poly( -aminoester) Poly(4-hydroxy-L-proline ester) PHP Poly(allylamine) Poly(a-[4-aminobuty]]-L-glycolic acid) PAGA Poly(D,L-lactic-co-glycolic acid) PLGA Poly(N -ethyl-4-vinylpyridinium bromide) Poly(phosphazene)s PPZ Poly(phosphoester)s PPE Poly(phosphoramidate)s PPA Poly(N -2-hydroxypropylmethacrylamide) pHPMA Poly (2-(dimethylamino)ethyl methacrylate) pDMAEMA Poly(2-aminoethyl propylene phosphate) PPE-EA Chitosan Galactosylated chitosan N -Dodacylated chitosan Histone Collagen Dextran-spermine D-SPM

[0968] n an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

[0969] In an embodiment, liposomes are used for delivery, e.g., to blood or bone marrow, e.g., as a way of targeting hematopoietic stem cells (HSCs) and progenitors. For example, long-term treatment can be enabled by direct delivery using liposomes for conditions where obtaining HSCs is difficult (e.g., HSCs are not stable or HSCs are rare). These conditions can include, e.g., sickle cell anemia, Fanconi anemia, and aplastic anemia. In an embodiment, liposomes are used for delivery to localized specific tissues, e.g., to liver or lung, via intravenous delivery or via localized injection to target organ or its blood flow. For example, long-term treatment can be enable to concentrate effect in that specific orgari or tissue type. These conditions can include urea cycle disorders, alpha- 1-anti-trypsin or cystic fibrosis.

[0970] In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bateriophase (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequencesn and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the "empty" particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the target patient (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes - patient derived membrane-bound nanovescicle (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).

[0971] In an embodiment, delivery of Cas by nanoparticles in the bone marrow is an in vivo approach to curing blood and immune diseases.

[0972] In an embodiment, the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component described herein is delivered by nucleofection. For example, Nucleofector™ (Lonza Cologne AG) is a transfection technology that can be used for delivery to primary cells and difficult-to-transfect cell lines. It is a non-viral method based on a combination of electrical parameters and cell-type specific solutions. It allows transfected nucleic acids to directly enter the nucleus (e.g., without relying on cell division for the transfer of nucleic acids into the nucleus), providing the ability to transfect non-dividing cells, such as neurons and resting blood cells. n an embodiment, nucleofection is used as an ex vivo delivery method.

[0973] In an embodiment, the components of a Cas system, e.g , the Cas9 molecule component and the gRNA molecule component described herein is delivered by methods utilizing endogenous receptor-mediate transporters, e.g., antibody-based molecular Trojan Horses (ArmaGen). Such methods can allow for non-invasive delivery of therapeutics to locations that are otherwise difficult to reach, e.g., brain (e.g., to cross blood brain barrier (BBB), e.g., via endogenous receptor-mediated transport processes). / [0974] In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the compoments of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule compoment, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g, an RNA molecule described herein.

XIII. Bi- oda or Differential Delivery of Components

[0975] Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety. [0976] In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery, that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, template nucleic acid, or payload. E.g., the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.

[0977] Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno associated virus or lentivirus, delivery.

[0978] By way of example, the components, e.g., a Cas9 molecule and a gRNA molecule, can be delivered by modes that differ in terms of resulting half life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA molecule can be delivered by such modes. The Cas9 molecule component can be delivered by a mode which results in less persistence or less exposure of its to the body or a particular compartment or tissue or organ.

[0979] More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.

[0980] n an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property. [0981] In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

[0982] In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmcokinetic property, e.g., distribution, persistence or exposure.

[0983] In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.

[0984] n an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.

[0985] In an embodiment, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gR As are incapable of acting in isolation. The second component, a Cas9 molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 molecule/gRNA molecule complex is only present and active for a short period of time.

[0986] Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.

[0987] Use of differential delivery modes can enhance performance,' safety and efficacy. For example, the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part deliveiy system can alleviate these drawbacks.

[0988] Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In an embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.

[0989] When the Cas9 molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.

XIV. Targeting of Genomic Signatures

[0990] Cas9 molecules, gRNA molecules, and in particular, Cas9 molecule/gRNA molecule complexes, can be used to target a cell by virtue of sequence specific interaction with a target nucleic acid comprising a selected genomic signature. This provides for targeted destruction of cells having a selected genomic signature. Method and compositions disclosed herein can be used to treat disorders characterized by a selected genomic signature, e.g., a genomic signature present in the germline or a genomic signature that arise as a result of a sporadic or somatic change in the genome, e.g., a germline or acquired mutation in a cancer cell, a viral infection, or other germline or acquired changes to the genome.

[0991] While not wishing to be bound by theory, it is believed that complementarity between the targeting domain of a gRNA molecule and the target sequence of a target nucleic acid mediates target sequence-specific interaction of the Cas9 molecule/gRNA molecule complex with the target sequence. This allows targeting of specific sequences or genomic signatures, e.g., rearrangements, e.g., translocations, insertions, deletions, and inversions, and other mutations. A Cas9 molecule/gRNA molecule complex can be used to target specific sequence, e.g., mutations, that are genriline, mitochondrial, or somatic. Depending on the Cas9 molecule/gRNA molecule complex used, specific editing, the delivery of a payload, or both, can be effected. In an embodiment, both cleavage and delivery of a payload is effected.

[0992] In an embodiment, the Cas9 molecule/gRNA molecule complex that promotes cell death upon recognition of its target genomic sequence. In an embodiment, an eaCas9 molecule/gRNA molecule complex cleaves the target nucleic acid. In an embodiment, it does not deliver a payload. While not wishing to be bound by theory is it believed that endogenous cellular elements, e.g., elements of the DNA damage apoptosis signaling cascade promote apoptosis in these embodiments.

[0993] In an embodiment, an eaCas9 molecule/gRNA molecule complex cleaves the target nucleic acid and delivers a payload. The payload can comprises a compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade. In an embodiment, a second Cas9 molecule/gRNA molecule complex is used to deliver a payload comprising a second compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade. The Cas9 molecule/gRNA molecule complex that delivers the second payload can comprise an eiCas9 molecule or an eaCas9 molecule. An additional, e.g., third or fourth, Cas9 molecule/gRNA molecule complex, can be used to deliver additional payload, e.g., an additional compound that inhibits growth or cell division, or promotes apoptosis, e.g., an additional element of the DNA damage apoptosis signaling cascade promote.

[0994] n an embodiment, the Cas9 molecule/gRNA molecule complex delivers a payload comprising a compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade, but does not cleave the target nucleic acid. While not wishing to be bound by theoiy is it believed that endogenous cellular elements, e.g., elements of the DNA damage apoptosis signaling cascade promote apoptosis in these embodiments.

[0995] Exemplary compounds that inhibit growth or cell division, or promote apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade, are described herein, e.g., in Table

XIV- 1. Table XlV-1

ATM kinases (double-strand breaks) ATR kinases (single-strand breaks) RF-C related protein (RAD 17) The 9-1-1 Complex: RAD1, RAD9, and HUS1 Checkpoint proteins CH 1, CHK2 P53 ZIP Kinase (ZIPK) Fast Death-Domain Associated Protein XX (DAXX) Promyelocytic leukemia protein (PML) Apoptosis-inducing factor (AIF) Caspase-activated DNAse (CAD) (in the absence of its inhibitor ICAD)

[0996] In an embodiment, a Cas9 molecule/gRNA molecule complex targets a sequence that includes or is near the breakpoint of a rearrangement, e.g., a translocation, inversion, insertion, or deletion. In an embodiment, the rearrangement confers unwanted properties, e.g., unwanted proliferation, on the cell. In an embodiment, the cell harboring the rearrangement is a cancer

cell. In an embodiment, the rearrangement comprises a kinase gene and results in unwanted, increased, or constitutive expression of the kinase activity. In an embodiment, the rearrangement disrupts the expression of a tumor suppressor.

[0997] In an embodiment, the Cas9 molecule/gRNA molecule complex:

specifically targets, and e.g., cleaves, the genome of a cell comprising a rearrangement, e.g., by targeting a mutation, e.g., a breakpoint or junction of a rearrangement; or

targets, e.g., for cleavage or payload delivery, a nucleotide sequence within 200, 100, 150, 100, 50, 25, 10, or 5 nucleotides of a mutation, e.g., a rearrangement breakpoint.

[0998] The invention includes a method of manipulating a cell comprising a genomic signature, comprising: administering a Cas9 molecule/gRNA molecule complex that targets said genomic signature, thereby manipulating said cell.

[0999] In an embodiment, manipulating comprises inhibiting the growth or division of, or killing, said cell. [1000] In an embodiment, said cell is a cancer cell or cell having a viral infection.

[1001] n an embodiment, the method comprises treating a subject, e.g., a human subject, for a disorder characterized by a cell having said genomic signature, e.g., a cancer or a viral infection.

[1002] In an embodiment, a Cas9 molecule/gRNA molecule complex disrupts a rearrangement, e.g., by introduction of a stop codon from a template nucleic acid, e.g., a stop codon is inserted into a fusion protein, e.g., a fusion protein comprising kinase activity.

[1003] The invention includes a method of treating a cancer having a translocation of a kinase gene to a non-kinase gene, which places the kinase domain under the control of the non-kinase gene control region comprising:

administering a Cas9 molecule/gRNA molecule complex that targets the translocation. In an embodiment, the control region, e.g., the promoter, or the coding sequence, of the kinase translocation, is edited to reduce expression.

XV. Combination Therapy

[1004] The Cas9 molecules, gRNA molecules, and in particular, Cas9 molecule/gRNA molecule complexes, can be used in combination with a second therapeutic agent, e.g., a cancer drug. n some embodiments, the second therapeutic agent (e.g., a cancer drug) and the Cas9 molecule, gRNA molecule, and in particular, Cas9 molecule/gRNA molecule complex target different (e.g., non-overlapping) pathways. In other embodiments, the second therapeutic agent (e.g., a cancer drug) and the Cas9 molecule, gRNA molecule, and in pailicular, Cas9 molecule/gRNA molecule complex target a same or overlapping pathway.

[1005] Exemplary combination therapies include, e.g.:

mTOR inhibitors (e.g., Temsirolimus (Torisel®) or Everolimus (Afinitor®)) together with a AKT-specific Cas9/gRNA molecule; - Tyrosine kinase inhibitors such as Imatinib mesylate (Gleevec®); Dasatinib (Sprycel®); Bosutinib (Bosulif®); Trasruzumab (Herceptin®); Pertuzumab (Perjeta™); Lapatinib (Tykerb®); Gefitinib (Iressa©); Erlotinib (Tarceva®) together with a HDAC-specific Cas9/gRNA molecule; and Any chemotherapeutic agent together with one or more Cas9/gRNAs against multidrug resistance genes such as MDR1 gene.

XVI. Treatment of genetic disorder, e.g., Duchenne muscular dystrophy (DMD)

[1006] In another aspect, the invention features, a method of altering a cell, e.g., reducing or abolishing the effect of a genetic signature, e.g., a stop codon, e.g., a premature stop codon. The method comprises contacting said cell with:

a Cas9 molecule/gRNA molecule complex that cleaves at or upstream from the genetic signature, e.g., a premature stop codon,

thereby altering the cell.

[1007] While not wishing to be bound by theory it is believed that, in an embodiment, cleavage and subsequent exonuclease activity, and non-homologous end joining results in an altered sequence in which the genetic signature, e.g., a premature stop codon is eliminated, e.g., by being placed in a different frame. In an embodiment, the same series of events restores the proper reading frame to the sequence that follows the signature, e.g., premature stop codon.

[1008] When the method is earned out to correct a frameshift mutation in order to remove a premature stop codon, repair can be earned out at various sites in the DNA. One may direct cleavage at the mutation, thereby correcting the frameshift entirely and returning the protein to its wild-type (or nearly wild-type) sequence. One may also direct cleavage at or near the premature stop codon, so that all (or nearly all) amino acids of the protein C-terminal of the codon where repair was effected are wild-type. In the latter case, the resulting protein may have one or more frameshifted amino acids between the mutation and the repair site; however the protein may still be functional because it is full-length and has wild-type sequence across most of its length.

[1009] A genetic signature is a particular DNA sequence at a particular portion of the genome, that causes a phenotype (such as a genetic disease or a symptom thereof)- For instance, the genetic signature may be a premature stop codon that prevents expression of a protein. In this scenario, the premature stop codon can arise from a mutation that directly creates a stop codon, or from a mutation that causes a frameshift leading to a premature stop codon being formed downstream. A genetic signature may also be a point mutation that alters the identity of an important amino acid in a protein, disrupting the protein's function.

[1010] In an embodiment, the Cas9 molecule/gRNA molecule complex mediates a double stranded break in said target nucleic acid.

[1011] In certain embodiments, the genetic signature, e.g., a premature stop codon, results from a point mutation, an insertion, a deletion, or a rearrangement. In some embodiments, a mutation causes a frameshift, resulting in a genetic signature, e.g., a premature stop codon downstream of the mutation..

[1012] In certain embodiments, the premature stop codon is within the target nucleic acid. In other embodiments, the target nucleic acid is upstream of the premature stop codon. The mutation may be upstream of the target nucleic acid, within the target nucleic acid, or downstream of the target nucleic acid.

[1013] In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the mutation. In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the genetic signature, e.g., a premature stop codon.

[1014] In certain embodiments, the Cas9 molecule/gRNA molecule complex mediates exonuclease digestion of the target nucleic acid. In certain embodiments, the Cas9

molecule/gRNA molecule complex removes 1, 2, 3, 4, or 5 nucleotides at the double stranded break.

[1015] In some embodiments, the double stranded break is resolved by non-homologous end joining.

[1016] In some embodiments the mutation and/or genetic signature, e.g., premature stop codon is in the dystrophin gene, e.g., in exon 51, or in the intron preceding or following exon 51. The premature stop codon may also be caused by a mutation in the dystrophin gene at one or more of codons 54, 645, 773, 3335, and 3340. In some embodiments, the premature stop codon in the dystrophin gene results from a deletion of codons 2305 through 2366.

[1017] In some embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises contacting the cell with a nucleic acid encoding a Cas9 molecule. In certain embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises transfecting the cell with a nucleic acid, e.g., a plasmid, or using a viral vector such as adeno- associated virus (AAV).

[1018] n certain embodiments, the method results in increased levels of the protein in which the genetic signature, e.g., a premature stop codon, was previously located. For instance, protein levels (e.g., dystrophin levels) may be increased by at least 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in a cell or in a tissue. In some embodiments, the method results in increased levels of the mRNA in which the premature stop codon was previously located, for instance by preventing the mRNA from undergoing nonsense-mediated mRNA decay.

[1019] In some embodiments, one or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation are located in the dystrophin gene (which is mutated in DMD). One or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation may also be located in the COL7A1 gene (mutated in type VH-associated dystrophic epidermolysis bullosa), the FKTN gene (mutated in Fukuyama congenital muscular dystrophy), the dysferlin gene (mutated in limb-girdle muscular dystrophy type 2B), the CFTR gene (mutated in cystic fibrosis), HEXA (mutated in Tay-Sachs disease), the IDS gene (mutated in Hunter syndrome), the FVUI gene (mutated in hemophilia), the IDUA gene (mutated in Hurler syndrome), the PPT 1gene (mutated in infantile neuronal ceroid lipofuscinosis), a tumor suppressor such as the ATM gene (mutated in cancers like gliomas and B-Cell Chronic Lymphocytic Leukemia), RP2 (mutated in X-linked retinitis pigmentosa), the CTNS gene (mutated in nephropathic cystinosis), and the AVPR2 gene (mutated in Congenital nephrogenic diabetes insipidus).

[1020] In some embodiments, the method is perfoiTned in cultured cells. In some embodiments, the method further comprises administering the cell to a patient. The cell may be, for example, an induced pluripotent stem cell, a bone marrow derived progenitor, a skeletal muscle progenitor, a CD133+ cell, a mesoangioblast, or a MyoD-transduced dermal fibroblast.

[ 1021 ] In some embodiments, the method comprises contacting the cell with a template nucleic acid under conditions that allow for homology-directed repair between the target nucleic acid and the template nucleic acid to correct the mutation or the premature stop codon. [1022] In another aspect, the invention features a method of treating a human subject having a disorder associated with a genetic signature, e.g., premature stop codon, e.g., DMD, comprising providing to the human subject:

1) a Cas9 molecule/gRNA molecule complex that cleaves at or upstream from the premature stop codon or

2) a cell that has been contacted with such complex,

thereby treating the subject.

[1023] n an embodiment,the Cas9 molecule/gRNA molecule complex mediates a double stranded break in said target nucleic acid.

[1024] In certain embodiments, genetic signature, e.g., premature stop codon results from a point mutation, an insertion, a deletion, or a rearrangement. In some embodiments, a mutation causes a frameshift, resulting in a premature stop codon downstream of the mutation.

[1025] In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the mutation. In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the premature stop codon.

[1026] In certain embodiments, the genetic signature, e.g., premature stop codon is within the target nucleic acid of the Cas9 molecule/gRNA molecule complex. In other embodiments, the target nucleic acid is upstream of the genetic signature, e.g., premature stop codon. The mutation may be upstream of the target nucleic acid, within the target nucleic acid, or downstream of the target nucleic acid.

[1027] In certain embodiments, the Cas9 molecule/gRNA molecule complex mediates exonuclease digestion of the target nucleic acid. In certain embodiments, the Cas9 molecule/gRNA molecule complex removes 1, 2, 3, 4, or 5 nucleotides at the double stranded break.

[ 028] In some embodiments the double stranded break is resolved by non-homologous end joining.

[1029] In some embodiments the mutation and/or genetic signature, e.g., premature stop codon is in the dystrophin gene, e.g., in exon 51, or in the intron preceding or following exon 51. The premature stop codon may also be caused by a mutation in the dystrophin gene at one or more of codons 54, 645, 773, 3335, and 3340. In some embodiments, the premature stop codon in the dystrophin gene results from a deletion of codons 2305 through 2366.

[1030] In some embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises contacting the cell with a nucleic acid encoding a Cas9 molecule. In certain embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises transfecting the cell with a nucleic acid, e.g., a plasmid, or using a viral vector such as aderio- associated virus (AAV).

[1031] In certain embodiments, the method results in increased levels of the protein in which the genetic signature, e.g., premature stop codon was previously located. For instance, protein levels (e.g., dystrophin levels) may be increased by at least 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in a cell or in a tissue. In some embodiments, the method results in increased levels of the mRNA in which the premature stop codon was previously located, for instance by preventing the mRNA from undergoing nonsense-mediated mRNA decay.

[1032] In some embodiments, one or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation are located in the dystrophin gene (which is mutated in DMD). One or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation may also be located in the COL7A1 gene (mutated in type VH-associated dystrophic epidermolysis bullosa), the FKTN gene (mutated in Fukuyama congenital muscular dystrophy), the dysferlin gene (mutated in limb-girdle muscular dystrophy type 2B), the CFTR gene (mutated in cystic fibrosis), HEXA (mutated in Tay-Sachs disease), the IDS gene (mutated in Hunter syndrome), the FVIII gene (mutated in hemophilia), the IDUA gene (mutated in Hurler syndrome), the PPT1 gene (mutated in infantile neuronal ceroid lipofuscinosis), a tumor suppressor such as the ATM gene (mutated in cancers like gliomas and B-Cell Chronic Lymphocytic Leukemia), RP2 (mutated in X-linked retinitis pigmentosa), the CTNS gene (mutated in nephropathic cystinosis), and the AVPR2 gene (mutated in Congenital nephrogenic diabetes insipidus).

[1033] In some embodiments, the method is performed in cultured cells. In some embodiments, the method further comprises administering the cell to a patient. T e cell may be, for example, an induced pluripotent stem cell, a bone marrow derived progenitor, a skeletal muscle progenitor, a CD133+ cell, a mesoangioblast, or a MyoD-transduced dermal fibroblast.

[1034] In some embodiments the method comprises contacting the cell with a template nucleic acid under conditions that allow for homology-directed repair between the target nucleic acid and the template nucleic acid to correct the mutation or the premature stop codon.

[1035] In some embodiments, the subject has a disorder selected from Duchenne Muscular Dystrophy (DMD), collagen type VH-associated dystrophic epidermolysis bullosa, Fukuyama congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B, cystic fibrosis, lysosomal storage disorders (such as Tay-Sachs disease, Hunter syndrome, and nephropathic cystinosis), hemophilia, Hurler syndrome, infantile neuronal ceroid lipofuscinosis, X-linked retinitis pigmentosa (RP2), cancers (such as gliomas and B-Cell Chronic Lymphocytic Leukemia), and Congenital nephrogenic diabetes insipidus.

XVII. Treatment of disorders characterized by lack of mature specialized cells, e.g., impaired hearing, with loss of hair cells, supporting cells, or spiral ganglion neurons; or for diabetes, with loss of beta islet cells

[1036] In another aspect, the invention features, a method of altering a cell, e.g., to promote the development of other mature specialized cells, e.g, in regeneration therapy. For example, proliferation genes can be upregulated and/or checkpoint inhibitors can be inhibited, e.g., to drive down one or more differenation pathways.

[1037] In an embodiment, the method includes induction of proliferation and specified lineage maturation.

[1038] In an embodiment, the method comprises, e.g., for restoration or improvement of hearing, contacting said cell with:

a Cas9 molecule/gRNA molecule complex that up-regulates a gene that promotes the development of hair cells, or down-regulates a gene that inhibits the development of hair cells thereby altering the cell.

[1039] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that up- regulates a gene that promotes hair cell development. [1040] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that down- regulates a gene that inhibits hair growth.

[1041] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to up-regulate a gene that promotes hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that up- regulates a gene that promotes hair growth.

[ 42] n an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to down-regulate a gene that inhibits hair growth. n an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that down- regulates a gene that promotes hair growth.

[1043] In an embodiment, said cell is an iPS cell, a native hair cell progenitor, or a mature hair cell.

[1044] In an embodiment, the Cas9 molecule/gRNA molecule and modifies expression of a gene, e.g., by modifying the structure of the gene (e.g., by editing the genome) or by delivery of a payload that modulates a gene. In an embodiment, the gene is a transcription factor or other regulatory gene.

[1045] n an embodiment, for hair cell or other mature cell regeneration, the method includes one or more or all of the following:

contacting the cell with a Cas9 molecule/gRNA molecule complex that results in upregulation one or more of the following for cell proliferation: c-Myc, GATA3, Oct4, Sox2, Wnt, TCF3;

contacting the cell with a Cas9 molecule/gRNA molecule complex that results in downregulation one or more of the following for check point: CL2 BMP, Hes , Hes5, Notch, p27, Prox , TGF|3; and

contacting the cell with a Cas9 molecule/gRNA molecule complex that results in turning on a maturation pathway. For hair cells this would include one or more of the following: Atoh 1

(Malhl). Barhl 1, Gfil , Myo7a, p63, PAX2, P , Pou4f3 arid for neurons would include one or more of the following: NEFH, Neurodl, Neurogl , POU4F1 . [1046] In an embodiment, the method comprises generation of inner ear hair cells, outer ear hair cells, spiral ganglion neurons, and ear supporting cells.

[1047] n an embodiment, one or more growth factors can be modulated, e.g., upregulated, e.g., TPO can be upregulated for production of platelets and GCSF can be upregulated for production of neutrophils.

[1048] In another aspect, the invention provides altered cell described herein, e.g., in this Section XVII.

[1049] In another aspect, the invention features a method of treating impaired hearing. The method comprises administering to said subject, an altered cell described herein, e.g., in this section XVII. In an embodiment, the cell is autologous. In an embodiment, the cell is allogeneic. In an embodiment, the cell is xenogeneic.

[1050] In another aspect, the invention features a method of treating subject, e.g., for impaired hearing. The method comprises administering to said subject:

a Cas9 molecule/gRNA molecule complex that up-regulates a gene that promotes the growth of hair, or down-regulates a gene that inhibits the growth of hair thereby altering the cell.

[1051] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that up- regulates a gene that promotes hair growth.

[1052] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that down- regulates a gene that inhibits hair growth.

[1053] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to up-regulate a gene that promotes hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that up- regulates a gene that promotes hair growth.

[1054] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to down-regulate a gene that inhibits hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that down- regulates a gene that promotes hair growth. [1055] In an embodiment, the Cas9 molecule/gRNA molecule and modifies expression of a gene, e.g., by modifying the structure of the gene (e.g., by editing the genome) or by delivery of a payload that modulates a gene. In an embodiment, the gene is a.transcription factor or other regulatory gene.

[1056] In an embodiment, the method includes one or more or all of the following:

administering a Cas9 molecule/gRNA molecule complex that results in up-regulation one or more of the following: c-Myc, GATA3, Oct4, Sox2, Wnt, TCF3;

administering a Cas9 molecule/gRNA molecule complex that results in turning on a maturation pathway. For hair cells this would include one or more of the following: Atol 1 (Mathl), Barhll, Gfil, Myo7a, p63, PAX2, PAX8. Pou4f3 and for neurons would include one or more of the following: NEFH, Neurodl, Neurogl, POU4F1 .

Annexes are included as part of the application. INCORPORATION BY REFERENCE

[ 057] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

[1058] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While this invention has been disclosed with reference to. specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

BHLHE23 BHLHE40 j BHLHE41 \ C13orf38- SOHLH2 Obesity and metabolic syndrome, j CLOCK gliomas, Alzheimer's, infertility FERD3L j FIGLA i premature ovarian failure type 6 i HANDl : HAND2 j HELT HES1 j HES2 HES3 ; HES4 i HES5 \ HES6 \ HES7 i HEY1 : osteopenia and chondrocyte HEY2 hypertrophy in bone and glioblastoma HEYL Multiple cancers (regulating HIF1A angiogenesis) and ischemic disease i ID1 \ Pancreatic carcinoma : ID2 ID3 ID4 KIAA2018 LOC388553 \ LYL1 i MAX Hereditary pheochromocytoma i MESP1 j Spondylocostal dysostosis, autosomal \ MESP2 recessive 2 \ MITF MLX MLXIP MLXIPL j MNT i MSC i MSGN1 MXD1 Acute leukemia, melanoma MXD3 i MXD4 MXI1 MYC Hematopoietic tumors, leukemias and lymphomas, including Burkitt lymphoma MYCL1 MYCN Neuroblastoma MYF5 MYF6 Myopathy, centronuclear Squamous cell carcinomas and \ MYOD1 malignant melanomas i MYOG i NEUROD1 Type 2 diabetes i NEUROD2 NEUROD4 i NEUROD6 j NEUROG1 j NEUROG2 NEUROG3 Diarrhea 4, malabsorptive, congenital j NHLH1 ; NHLH2 NPAS1 i NPAS2 NPAS3 Schizophrenia and mental retardation \ NPAS4 OLIG1 ; NSCLC and mutiple sclerosis Alzheimer's disease, Down syndrome, \ OLIG2 : pilocytic astrocytoma OLIG3 PTF1A SCXA SCXB SIM1 j SOHLH1 j SOHLH2 SREBF1 SREBF2 TALI \ ALL ; TAL2 i TCF12 j TCF15 j TCF21 \ TCF23 i TCF3 TCF4 j TCFL5 TFAP4 i TFE3 j Renal cell carcinoma, papillary, 1 TFEB TFEC TWIST1 Saethre-Chotzen syndrome. TWIST2 j USF1 Familial combined hyperlipidemia i USF2 C/EBP Regulate cell differentiation, ! Cutaneous squamous cell carcinoma \ ATF3 growth, survival, and and other cancers inflammation i ATF7 i Breast cancer BATF3 i CEBPA i AML CEBPB CEBPD CEBPE i CEBPG AML, non-bronchogenic carcinoma CREB5 XBP1 \ T-ALL (T-cell acute lymphoblastic leukemia),[25] CADASIL (Cerebral Autosomal-Dominant Arteriopathy with \ CBFB Sub-cortical Infarcts and Leukoencephalopathy), M S (Multiple

. Sclerosis), Tetralogy of Fallot, Alagille syndrome

\ CG-1 Calcium signaling by direct CAMPTAl, CAMPTA2: Cardia CAMTA1 binding of calmodulin. : hypertrophy, congenital ataxia i CAMTA2 EBF1 EBF1 throught 4: Hodgkin Lymphoma glioblastoma multiforme and gastric EBF2 carcinoma i EBF3 EBF4 COU P Involved in widely diverse i NR2F1 physiological functions, j NR2F2 including control of embryonic development, cell differentiation and NR2F6 homeostasi j Colon cancer CP2 , Progressive autosomal dominant GRHL1 hearing loss GRHL2 : GRHL3 j Hepatocellular carcinoma, Alzheimer's TFCP2 disease TFCP2L1 UBP1

CSD Cold shock domain proteins CARHSPl protect the cells when j CSDA Hepatocellular carcinoma, gastric cancer temperatures go below \ CSDC2 optimum growth i CSDEl temperatures LIN28A LIN28B YBX1 YBX2 i Male factor infertility CSL Universal transcriptional CSL effector of Notch signaling. Notch signaling is dysregulated in many i Prostate cancer cancers, and faulty notch signaling is implicated in many diseases CTF/NFI Functions both in viral DNA NFIA, NFIB, NFIC, NFIX: Lymphedema- replication and in the NFIA distichiasis, developmental verbal regulation of gene dyspraxia, autoimmune diseases expression Breast and head and neck adenoid NFIB cystic carcinoma NFIC \ NFIX CUT i cuxi : AML, major depressive disorder CUX2 ONECUT1 O ECUT2 ONECUT3 SATB1 Prostate cancer Isolated cleft palate and mental SATB2 retardation

Believed t o function as Defective testicular development and XY ; \ DMRT1 j transcription factors on i feminization downstream sex- DMRT2 i Gonadal dysgenesis and XY sex reversal determination genes i D T3 DMRTAl \ D RTA2 \ DMRTB1 D TC2 E2F involved in the cell cycle E2F1 regulation and synthesis of : E2F2 : Involvement in most cancers DNA. Some are activators \ E2F3 and others are suppressors E2F4 i E2F5 E2F6 i E2F7 ! E2F8 i TFDP1 TFDP2 \ TFDP3 involved in widely diverse Ecdystd NR1H2 Preeclampsia receptor physiological functions, including control of NR1H3 embryonic development, Atherosclerosis

Anterior segment mesenchymal \ FOXE3 ! dysgenesis Alveolar capillary dysplasia with \ FOXF1 i misalignment of pulmonary veins i FOXF2 I FOXG1 : Rett syndrome, congenital variant i FOXH1 FOXI1 i Enlarged vestibular aqueduct \ FOXI2 FOXJ1 FOXJ2 FOXJ3 i FOXK1 FOXK2 FOXL1 i Blepharophimosis-ptosis-epicanthus \ FOXL2 i inversus syndrome : FOX 1 Cancer T-cell immunodeficiency, congenital \ FOXN1 i alopecia, and nail dystrophy FO FO 3 \ FOXN4 \ FOXOl Autoimmune diseases FOX03 FOX04 FOX06 FOXP1 FOXP2 Developmental verbal dystraxia IPEX (immunodysregulation polyendocrinopathy enteropathy X- i FOXP3 linked syndrome) and autoimmune i diseases \ FOXP4 i FOXQl FOXR1 FO 2 FOXS1 KIAA0415 GCM Regulators of gliogensis GCMl severe congenital isolated GCM2 hypoparathyroidism. GCR Control of embryonic development, cell NR3C1 differentiation and homeostasis Generalized glucocorticoid resistance GTF2I Involvement in the GTF2I Williams-Beuren Syndrome morphological and neuro- GTF2IRD1 Williams-Beuren Syndrome developmental anomalies GTF2IRD2 Williams-Beuren Syndrome GTF2IRD2B Williams-Beuren Syndrome G Involved in the regulation of BBX patterns of anatomical CIC development HBP1 HMG20A HMG20B HMGB1 HMGB2 HMGB3 HMGXB3 HMGXB4 LEF1 PBRM1 PINX1 i Parkinson diseased, early onset P S1 SMARCE1 SOX1 Hepatocellular carcinoma \ Waardenburg-Shah and Waardenburg- \ SOX10 Hirschsprung disease SOX11 Epithlian ovarian cancer SOX12 i SOX13 SOX14 i SOX15 i SOX17 Hypotrichosis-lymphedem telangiectasia syndrome SOX2 Microphthalmia, syndromic 3 SOX21 Mental retardation, X-linked, with SOX3 isolated growth hormone deficiency SOX30 SOX4 SOX5 sox6 \ SOX7 \ SOX8 sox9 i Acampomelic campomelic dysplasia SRY Swyer syndorme and XX male syndrome : SSRP1 \ TCF7 \ TCF7L1 \ TCF7L2 TFAM i TOX i TOX2 TOX3 TOX4 UBTF UBTFL1 UBTFL2 WHSC1 Wolf-Hirschhorn syndrome HMGI/ HMGY Non-histone nuclear Cancer, fibrosis and autoimmune H GA1 proteins which constitute diseases important components chromatin structure. HMGA2 Liposarcoma Homeobox Over 200 different genes involved in development, ADNP differentiation and tumor ALX1 Axenfeld-Rieger syndrome suppression. Following branchiootorenal syndrome homeobox genes have a ALX3 coloboma genetic disease association; ALX4 combined pituitary hormone deficiency ALX4, ARX, HESX1, HOXA13, ARGFX congenital central hypoventilation LMX1B, MSX1, MSX2, OTX2, syndrome PAX2, PAX3, PAX6, PAX8, ARX congenital fibrosis of the extraocular PHOX2A, PHOX2B, PITX2, BARHL1 muscles POU3F4, PROP1, SHOX, SIX1, BARHL2 congenital hypothyroidism SIX3, SIX5, TGIF1, and ZEB2. BARX1 craniofaciai-deafness-hand syndrome enlarged parietal foramina BARX2 hand-foot-genital syndrome BSX Langer mesomelic dysplasia CDX1 Leri-Weill dyschondrosteosis microphthalmia CDX2 Mowat-Wilson syndrome CDX4 nail-patella syndrome DBX1 neuroblastoma nonsyndromic deafness DBX2 nonsyndromic holoprosencephaly DLX1 Partington syndrome DLX2 Potocki-Shaffer syndrome renal coloboma syndrome DLX3 septo-optic dysplasia DLX4 Turner syndrome DLX5 Waardenburg syndrome Wilms tumor, aniridia, genitourinary DLXG anomalies, and mental retardation D BX1 syndrome DPRX Wolf-Hirschhorn syndrome X-linked infantile spasm syndrome DRGX X-linked lissencephaly with abnormal DUX4L2 genitalia DUX4L3

DUX4L5

DUX4L6

DUXA

EMX1

EMX2 E

EN2

ESX1

EVX1

EVX2 GBX1

GBX2

GSC

GSC2

GSX1

GSX2 HDX Growth hormone deficiency with HESX1 pituitary anomalies HHEX

HLX

HMBOX1 HMX1

HMX2 HMX3

HNF1A MODY HNF1B MODY HOMEZ HOPX HOXA1

HOXA10 HOXA11

HOXA13 Microtia, hearing impairment, and cleft HOXA2 palate

HOXA3 HOXA4

HOXA5 HOXA6 HOXA7 HOXA9 HOXB1

HOXB13 HOXB2 HOXB3 HOXB4

HOXB5 ' :

HOXB6 HOXB7

HOXB8

HOXB9 HOXC10

HOXC11

HOXC12

HOXC13 HOXC4

HOXC5

HOXC6 HOXC8

HOXC9

HOXD1 HOXD10 i Vertical talus, congenital HOXD11

HOXD12

HOXD13 Brachydactyly type D HOXD3

HOXD4

HOXD8

HOXD9

IRX1

IRX2

IRX3

I X4

IRX5 IRX6

ISL1

ISL2

ISX

LASS2

LASS3 LASS4

LASS5

LASS6

LBX1 LBX2

LEUTX

LHXl

LHX2

LHX3 Pituitary disease LHX4

LHX5

LHX6

LHX8

LHX9 LMX1A LMX1B

MEIS1 MEIS2 MEIS3 ME0X1 MEOX2 MIXL1 MNX1

MSX1 Witkop syndrome \ MSX2 Craniosynostosis, type 2 NANOG NKXl-1 i NKX2-1 i NKX2-2 j Chorea, hereditary benign

NKX2-3 NKX2-4 NKX2-5 NKX2-6 : Persistent truncus arteriosus NKX2-8 NKX3-1 NKX3-2 NKX6-1 NKX6-2 NKX6-3 NOBOX NOTO OTP PBXl PBX2

PBX3

PBX4 \ PDX1 Fibrosis of extraocular muscles, PHOX2A i congenital, 2 PHOX2B j PITX1 i Clubfoot, congenital PITX2 i Iridogoniodysgenesis, type 2 Anterior segment mesenchymal j PITX3 i dysgenesis PKNOX1 \ PKNOX2 \ PROP1 \ PRRXl

PRRX2 i RAX \ RAX2 j RHOXF1 RHOXF2 \ RHOXF2B

SEBOX 1 SHOX j SHOX2 i SIX1 i Brachiootic syndrome 3 \ SIX2 SIX3 j Holoprosencephaly-2 i SIX4 \ SIX5 Branchiootorenal syndrome 2 i Microphthalmia, isolated, with cataract SIX6 \ 2 j TGIFl i TGIF2 TGIF2LX TGIF2LY TLXl

TLX2 TLX3 UNCX VAX1 VAX2 i VENTX i vsxi

\ VSX2

ZEB1

i ZFHX3 ZFHX4 Ptosis, congenital

ZHX1

: ZHX2

ZHX3 HSF Activators of heat shock ut iple myeloma and other malignant \ HSF1 proteins i cancers : HSF4 I Congenital and age related catarats HSFX1 HSFX2 \ HSFY1 HSFY2

LCOR Involved in development i LCOR and metabolism regulation LCORL IRF : Important in the regulation IRF1 AML \ of interferons in response t o i IRF2 Pancreatic cancer I infection by virus and in the regulation of interferon- i IRF3 Increased susceptibility t o hepatitis virus diffuse large B-cell and follicular inducible genes \ IRF4 lymphomas. IRF5 Melanoma Woude syndrome, popliteal pterygium 6 syndrome and non-syndromic orofacial ; cleft type 6 i IRF7 ! Systemic lupus erythematosus. IRF8 i Dendritic-cell immunodeficiency i IRF9 M BD genomic imprinting, BAZ2A transposon and BAZ2B chromosome X inactivation, MBD1 Pancreatic cancer differentiation, and cancer MBD2 i MBD3 BD4 i Rheumatoid arthritis MECP2 Rett Syndrome i SETDB1 SETDB2 M H1 Inflammation, cancer, and i SMAD 1 through 7 and SMAD 9: i metabolic disorders S AD1 .i Colorectal, ovarian, NSCLC and prostate cancer, lupus S A D2 SMAD3 SMAD4 SMAD5 SMAD6 SMA07 SMAD9 ; MYB Regulator of proliferation, i CCDC79 i ALL differentiation and cell fate/ CDC5L 25 different genes, yet the ; D TF1 i NSCLC most representative is MYB i DNAJCl i Ovarian cancer i DNAJC2 : MIER3 MYB i Breast and prostate cancer MYBL1 \ MYBL2 MYSMl NCORl i NCOR2 i RCOR1 i Advanced osteoarthritic chondrocytes. i RCOR2 RCOR3 i SMARCC1 i SMARCC2 Gastric and colorectal cancer SNAPC4 j TADA2A TADA2B : TERF1 ! NSCLC ! TERF1P2 TERF2 TTF1 ZZZ3 Hematopoiesis and NDT80/PhoG \ Cllorfa development of bone tissue NF-YA \ NFYA NF-YB/C NFYB NFYC Nrfl i NRF1 Nuclear orphan Involved in widely diverse NR4A1 : Gastric cancer ! receptor physiological functions, including control of \ NR4A2 Rheumatoid arthritis embryonic development, cell differentiation and NR4A3 i Extraskeletal myxoid chondrosarcomas homeostasis. Estrogen Control of embryonic development, cell i Estrogen resistance, familial breast receptor ESR1 differentiation and cancer, migraine, myocardial infarction homeostasis Other nuclear Hepatocellular carcinoma and X-linked receptor NR0B1 congenital adrenal hypoplasia and hypogonadotropic hypogonadism

Fibrolamellar carcinoma and NR0B2 hepatocellular carcinoma.

Control development and P53 TP53 Large variety of cancers differentiation and play a role in tumor suppression : Ectodermal dysplasia, and cleft lip/palate syndrome 3 (EEC3); split- hand/foot malformation 4 (SHFM4); ankyloblepharon-ectodermal defects- TP63 cleft lip/palate; ADULT syndrome (acro- dermato-ungual-lacrimal-tooth); limb- mammary syndrome; Rap-Hodgkin syndrome (RHS) TP73 myelodysplastic syndrome. PAX critical role in the formation Cervical cancer, vertebral PAX1 of tissues and organs during malformations, Klippel-Feil syndrome embryonic development Optic nerve colobomas and renal PAX2 hypoplasia Waardenburg syndrome, craniofacial- PAX3 deafness-hand syndrome, and alveola rhabdomyosarcoma MODY, early onset-age type 2 diabetes, PAX4 melanoma PAX5 Lymphomas ocular disorders such as aniridia and PAX6 Peter's anomaly PAX7 Alveolar rhabdomyosarcoma Thyroid dysgenesis, thyroid follicular PAX8 carcinomas and atypical follicular thyroid adenomas PAX9 Oligodontia PC4 Replication, DNA repair and SUB1 transcription POU Wide variety of functions, all POU Genes : Aautoimmune arthritis, of which are related t o the asthma, septic shock, lung fibrosis, POU1F1 function of the glomerulonephritis, atherosclerosis, and neuroendocrine system and AIDS the development of an POU2F1 organism POU2F2 POU2F3 Pituitary hormone deficiency, POU3F1 combined, 1 POU3F2 POU3F3 POU3F4 Deafness, X-linked 2 POU4F1 POU4F2 POU4F3 Deafness, autosomal dominant 15 POU5F1 POU5F1B POU6F1 \ POU6F2 involved in cell proliferation, : PPAR receptor PPARA cell differentiation and in \ CLL, bladder cancer immune and inflammation PPARD : Colorectal cancer responses. j PPARG ; Thyroid cancer Progesterone Plays a central role in reproductive events : receptor associated with the \ PGR establishment and maintenance of pregnancy i Breast and endometrial cancer Proxl Essential for the PROXi Multiple cancers development and maintenance of lymphatic \ PROX2 vasculature Retinoic acid Control of embryonic ESRRA development, cell receptor NR2C1 differentiation and i NR2C2 homeostasis NR2E1 i NR5A1 NR5A2 j NR6A1 FX Regulate development and RFX1 Bardet-Biedl syndrome function of cilia : RFX2 RFX3 RFX4 RFX5 Bare lymphocyte syndrome, type II RFX6 i RF 7 i RFX8

RH D 10 different genes. One of the most important is NFKB: Autoimmune NFAT5 arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS

NFATC1 NFATC2 NFATC3 NFATC4 i Cancer, inflammatory and autoimmune NFKB1 diseases, septic shock, viral infection, and improper immune development Inflammatory events associated with NFKB2 autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, j atherosclerosis, and AIDS i REL j RELA j RELB RO receptor Control of embryonic \ NR1D2 development, cell RORA Autism differentiation and i RORB j Bipolar disorder homeostasis i RORG i Inflammatory auto immune Essential for osteoblastic Runt j RUNX1 i Different leukemia differentiation and skeletal morphogenesis \ RUNX3 ; Ranal cell carcinoma, psoriatic arthritis SAND j Autoimmune polyendocrinopathy with \ AIRE candidiasis and ectodermal dystrophy \ (APECED) i DEAF1 \ Colorectaladenocarcinomas GMEB1 \ GMEB2 i SP100 i Brain tumors ! SP110 i Tubercolosis, tubercolosis SP140 \ SP140L SRF Involved incell proliferation LOC729991- and differentiation \ EF2B MEF2A Coronary artery disease j MEF2B Parkinson disease and myotonic i EF2D i dystrophy Gastric cancer and hepatocellular \ SRF carcinoma STAT Regulates many aspects of STATs:Angiogenesis, enhanced tumor growth, survival and STAT1 survival and immunosuppression. differentiation in cells. \ STAT2 i STAT3 Hyper-lgE recurrent infection syndrome i STAT4 i STAT5A Growth hormone insensitivity with STAT5B i immunodeficiency STAT6 Involved in limb and heart T-box T-Box genes involved in: Alzheimer's, development i Parkinson's, ulnar-mammary syndrome \ EOMES chronic and congenital heart defects like Holt-Oram syndrome and obstructive coronary diseases

MGA i T Oral squamous cell carcinoma. i TBR1 \ TBX1 Conotruncal anomaly face syndrome TBX10 TBX15 \ Cousin syndrome i TBX18 TBX19 i TBX2 TBX20 TBX21 Cousin syndrome : TBX22 TBX3 i TBX4 Small patella syndrome \ TBX5 j TBX6 TEAD Essential for cardiac, i TEAD1 Sveinsson's chorioretinal atrophy skeletal, and smooth i TEAD2 development \ TEAD3 TEAD4 T F bZIP Important regulators of Angiomatoid fibrous histiocytoma and ATFl development such as clear cell sarcoma proliferation, differentiation \ ATF2 and transformation ATF4 ATF5 ATF6 ATF6B BACHl BACH2 i BATF BATF2 i Histiocytoma, angiomatoid fibrous, CREB1 : somatic CREB3 : Cerical cancer j CREB3L1 i CREB3L2 CREB3L3 CREB3L4 CREBL2 CREM i ! DBP DDIT3 FOS Multiple cancers FOSB : Multiple cancers FOSLl Multiple cancers i FOSL2 Multiple cancers i HLF i JDP2 JUN Breast and lung cancer

\ HNF4G j MODY, obesity ! A RG RXRA Acute promyelocyte leukemia ESRRG Breast cancer i RXRB type II vitamin D-resistant rickets TSC22 i Gliobastoma, slaivary gland and prostate cancer

i Inflammation, RA

Tub Associated with neuronal TUB differentiation and Obesity and sensorineural degradation development TULP1 i Retinitis pigmentosa TULP2 TULP3 TULP4 ZBTB Regulate lymphoid \ BCL6 i Diffuse large-cell lymphoma (DLCL) development and function j BCL6B Gastric cancer i GZF1 i HICl \ HIC2 \ MYNN PATZ1 ZBTB1 i ZBTB10 ZBTB11 ZBTB12 Skeletal defects, genital hypoplasia, and ZBTB16 ! mental retardation ZBTB17 ZBTB2 ZBTB20 i Hepatocellular carcinoma ZBTB24 j ZBTB25 ZBTB26 j ZBTB3 i ZBTB32 ZBTB33 ZBTB34 ZBTB37 ZBTB38 ZBTB4 ZBTB40 \ ZBTB41 ZBTB42 i ZBTB43 i ZBTB44 ZBTB45 ZBTB46 ZBTB47 ZBTB48 ZBTB49 ZBTB6 ZBTB7A ZBTB7B ZBTB7C ZBTB8A ZFP161 ZNF131 ZNF238 ZNF295 zf-BED Cllorf95 Chondroid lipoma \ ZBEDl i ZBED2 ZBED3 : ZBED4 zf-C2H2 Large family of transcription X-linked spinal, bulbar muscular atrophy BCL11A factors group together and prostate cancer because of the presence of \ BCL11B B-Cell malignancies zinc fingers in their BNC2 structure. Invoved with in regulating a multitude of i CTCF functions \ CTCFL \ E4F1 \ EG 1 Prostate and breast cancer Congenital hypomyelinating neuropathy and Charot-Marie-Tooth type 1 EGR3 EGR4 FEZF1 FEZF2 FIZ1 GF GFI1B GUI GLI2 Holoprosencephaly-9 GLI3 Polydactyly, postaxial, types Al and B GLI4 GLIS1 GLIS2 Nephronophthisis 7 Diabetes mellitus, neonatal, with GLIS3 congenital hypothyroidism GTF3A HINFP HIVEP1 HIVEP2 HIVEP3 HK 1 IKZF1 IKZF2 ZF3 IKZF4 IKZF5 INSM1 INSM2 KLF1 KLF10 KLF11 KLF12 KLF13 KLF14 KLF15 KLF16 KLF17 KLF2 KLF3 KLF4 KLF5 KLF6 KLF7 KLF8 KLF9 LOC100131539 LOC100132396 LOC100287841 MAZ ECO M MTF1 MZF1 OSR1 OSR2 OVOL1 OVOL2 OVOL3 PEG3 PLAG1 i Salivary gland pleomorphic adenomas PLAGL1 PLAGL2 PRD 1 PRDM10 PRDM12 PRDM13 PRDM14 PRDM15 PRDM16 PRDM2 j PRDM4 PRDM5 PRD 6 PRDM8 i PRDM9 RBAK REPIN1

REST Glioblastoma \ RREBl j SALL1 i SALL2 SALL3 i SALL4 j SCRT1 SCRT2

SNAI1 : Various cancers i SNAI2 SNAI3 i SP1 j Various cancers i SP2 ; SP3 j SP4 j SP5 i SPG SP7 j SP8 S 9 TRERFl \ TSHZ1 j TSHZ3 i VEZF1 wiz Ti Nephrotic syndrome and Wilms tumor YYl YY2 ZEB2 i ZFAT i ZFP1 j ZFP112 ZFP14 j ZFP2 ZFP28 ZFP3 \ ZFP30 ZFP37 ZFP41 ZFP42 i ZFP57 Diabetes mellitus, transient neonatal, 1 i ZFP62 j ZFP64 ZFP82 \ ZFP90 ZFP91 ZFPM1 \ ZFPM2 i Diaphragmatic hernia 3 i ZFX ZFY ZlCl j Isolated Dandy-Walker malformation \ ZIC2 Alobar holoprosencephaly Isolated congenitally uncorrected \ ZIC3 i transposition of the great arteries ZIC4 Isolated Dandy-Walker malformation \ ZIC5 ziKi ZI 2 ZI 3 i ZKSCAN1 ZKSCAN2 ZKSCAN3 ZKSCAN4 ZKSCAN5 ZNF10 ZNF100 ZNF101 i ZNF107 i ZNF114 i ZNF117 i ZNF12 ZNF121 ZNF124 ZNF132 ZNF133 i ZNF134 ZNF135 ! ZNF136 : ZNF138 : ZNF14 ZNF140 ZNF141 i Postaxial Polydactyly type A, bilateral ZNF142 ZNF143 ZNF146 ZNF148 j ZNF154 ! ZNF155 ZNF157 ZNF16 ZNF160 ZNF165 ZNF167 ZNF169 ZNF17 ZNF174 ZNF175 ZNF177 ZNF18 ZNF180 ZNF181 ZNF182 ZNF184 ZNF189 ZNF19 ZNF192 ZNF193 ZNF195 ZNF197 ZNF2 ZNF20 ZNF200 ZNF202 ZNF205 ZNF208 ZNF211 ZNF212 ZNF213 ZNF214 ZNF215 ZNF217 ZNF219 ZNF22 ZNF221 ZNF222 ZNF223 ZNF224 ZNF225 ZNF226 ZNF227 ZNF229 ZNF23 ZNF230 ZNF232 ZNF233 ZNF234 ZNF235 ZNF236 ZNF239 ZNF24 ZNF248 ZNF25 ZNF250 ZNF251 ZNF253 ZNF254 ZNF256 ZNF257 ZNF26 ZNF263 ZNF264 ZNF266 ZNF267 ZNF268 ZNF273 ZNF274 ZNF275 ZNF276 ZNF28 ZNF280D ZNF281 ZNF282 ZNF283 ZNF284 ZNF285 ZNF286A ZNF287 ZNF292 ZNF296 ZNF3 ZNF30 ZNF300 ZNF302 ZNF304 ZNF311 ZNF317 ZNF319 ZNF32 ZNF320 ZNF322A ZNF322B ZNF323 ZNF329 ZNF331 ZNF333 ZNF334 ZNF335 ZNF337 ZNF33A ZNF33B Z F34 ZNF341 ZNF343 ZNF345 ZNF347 ZNF35 ZNF350 ZNF354A ZNF354B i ZNF354C i ZNF358 i ZNF362 i ZNF366 ZNF367 ; ZNF37A ZNF382 ZNF383 i ZNF384 ZNF391 ZNF394 ZNF396 ZNF397 ZNF398 ZNF407

ZNF408 . Familial exudative vitreoretinopathy X-linked nonsyndromic intellectual ZNF41 deficit j ZNF410 j ZNF415 \ ZNF416 ZNF417 ZNF418 ZNF419 ZNF420 Joubert syndrome with oculorenal ZNF423 : defect ZNF425 ZNF426 ZNF429 ZNF43 ZNF430 ZNF431 ZNF432 ZNF433 ZNF434 ZNF436 ZNF439 ZNF44 ZNF440 ZNF441 ZNF442 ZNF443 ZNF444 ZNF445 ZNF446 ZNF449 ZNF45 ZNF451 ZNF454 ZNF4G0 ZNF461 ZNF467 ZNF468 ZNF469 i Brittle cornea syndrome ZNF470 ZNF471 ZNF473 ZNF479 ZNF48 ZNF480 ZNF483 ZNF484 ZNF485 ZNF486 ZNF490 ZNF491 ZNF492 ZNF493 ZNF496 ZNF497 ZNF498 ZNF500 ZNF501 ZNF502 ZNF506 ZNF507 ZNF510 ZNF512

\ ZNF574 : ZNF575 \ ZNF576 ZNF577 ZNF579 ZNF580 \ ZNF581 \ ZNF582 ZNF583 \ ZNF584 ZNF585A ZNF585B ZNF586 \ ZNF587 ZNF589 \ ZNF594 j ZNF596 ZNF597 ZNF599 ZNF600 ZNF606 \ ZNF607 ZNF610 ZNF611 i ZNF613 \ ZNF614 ZNF615 ZNF616 \ ZNF619 \ ZNF620 ZNF621 \ ZNF623 ZNF624 \ ZNF625 ZNF626 i ZNF627 ZNF628 j ZNFS29 i ZNF630 ZNF639 \ ZNF641 \ ZNF642 ZNF643 ZNF644 ZNF646 ZNF648 i ZNF649 ZNF652 ZNF653 ZNF655 ZNF658 ZNF660 ZNF662 ZNF66 ZNF665 ZNF667 ZNF668 ZNF669 ZNF670 ZNF671 ZNF672 X-linked nonsyndromic intellectual ZNF674 deficit ZNF675 ZNF676 ZNF677 ZNF678 ZNF679 ZNF680 ZNF681 ZNF682 ZNF683 ZNF684 ZNF687 ZNF688 ZNF689 ZNF69 ZNF691 ZNF692 ZNF696 ZNF697 ZNF699 ZNF7 ZNF70 ZNF700 ZNF701 ZNF705A ZNF705D ZNF705G ZNF707 ZNF708 ZNF709 ZNF71 ZNF710 X-linked nonsyndromic intellectual ZNF711 deficit ZNF713 ZNF714 ZNF716 ZNF717 ZNF732 ZNF736 ZNF737 ZNF74 ZNF740 ZNF746 ZNF749 ZNF75A ZNF75D ZNF76 ZNF763 ZNF764 ZNF765 ZNF766 ZNF768 ZNF77 ZNF770 ZNF771 ZNF772 ZNF773 ZNF774 ZNF775 ZNF776 ZNF777 ZNF778 ZNF780A ZNF780B X-linked nonsyndromic intellectual ZNF781 deficit ZNF800 ZNF805 ZNF808 ZNF81 ZNF813 ZNF814 ZNF816 ZNF821 ZNF823 ZNF827 ZNF83 ZNF831 ZNF836 ZNF837 ZNF84 ZNF841 ZNF844 ZNF845 ZNF846 ZNF85 ZNF852 ZNF860 ZNF865 ZNF878 ZNF879 ZNF880 ZNF90 ZNF91 ZNF92 ZNF93 ZNF98 ZNF99 ZSCAN1 ZSCAN10 ZSCAN12 ZSCAN16 ZSCAN18 ZSCAN2 ZSCAN20 ZSCA 21 ZSCAN22 ZSCAN23 ZSCAN29 ZSCAN30 ZSCAN4 ZSCAN5A ZSCAN5B ZSCAN5C ZXDA ZXDB ZXDC zf-C2HC L3MBTL1 L3MBTL4 YST2 YT 1 MYT1L ST18 zf-GATA GATA1 Congenital heart defects and cancer GATA2 Hypoparathyroidism, sensorineural GATA3 deafness, and renal dysplasia GATA4 GATA5 GATA6 GATAD1 GATAD2B MTA1 MTA2 MTA3 ERE TRPS1 ZGLP1 zf-LITAF-like C16orf5 LITAF zf-M Z PIAS1 PIAS2 PIAS3 PIAS4 RNF138 ZMIZ1 ZM I 2 zf-NF-Xl NFX1 NFXL1 ESR Control of embryonic ESR development, cell Estrogen resistance, familial breast differentiation and cancer, migraine, myocardial infarction homeostasis Table IX-2 (Annex IX-2) .

Disease Name t of Genes per Disease Gene Name

Duane-radial ray syndrome 1 SALL4 Achalasia-Addisonianism-Alacrimia syndrome 1 AAAS Charcot-Marie-Tpoth disease, axonal, type 2N 1 AARS

Saccharopinuria 1 AASS GABA-transaminase deficiency 1 ABAT HDL deficiency, type 2 1 ABCA1 Ichthyosis, harlequin 1 ABCA12 Ichthyosis, lamellar 2 1 ABCA12 Surfactant metabolism dysfunction, pulmonary, 3 1 ABCA3 Fundus flavimaculatus 1 ABCA4 Macular degeneration, age-related, 2 1 ABCA4 Retinal dystrophy, early-onset severe 1 ABCA4 Cholestasis, benign recurrent intrahepatic, 2 1 ABCB11 Cholestasis, progressive familial intrahepatic 2 1 ABCB11 Cholestasis, familial intrahepatic, of pregnancy 1 ABCB4 Cholestasis, progressive familial intrahepatic 3 1 ABCB4 Gallbladder disease 1 1 ABCB4 Anemia, sideroblastic, with ataxia 1 ABCB7 Pseudoxanthoma elasticum, forme fruste 1 ABCC6 Diabetes mellitus, noninsulin-dependent 1 ABCC8 Diabetes mellitus, permanent neonatal 1 ABCC8 Diabetes mellitus, transient neonatal 2 1 ABCC8 Hyperinsulinemic hypoglycemia, familial, 1 1 ABCC8 Hypoglycemia of infancy, leucine-sensitive 1 ABCC8 Cardiomyopathy, dilated, 10 1 ABCC9 Adrenoleukodystrophy 1 ABCD1 Gallbladder disease 4 1 ABCG8 Chanarin-Dorfman syndrome 1 ABHD5 ACAD9 deficiency 1 ACAD9 Acyl-CoA dehydrogenase, long chain, deficiency of 1 ACADL Acyl-CoA dehydrogenase, medium chain, deficiency of 1 ACADM Acyl-CoA dehydrogenase, short chain, deficiency of 1 ACADS VLCAD deficiency 1 ACADVL Osteochondritis dissecans, short stature, and early-onset osteoarthritis 1 ACAN Alpha-methylacetoacetic aciduria 1 ACAT1 1

Table IX-21781338.1 Lysosomal acid phosphatase deficiency 1 ACP2 Mast syndrome 2 ACP33 Mental retardation, X-linked nonspecific, 63 1 ACSL4 Myopathy, actin, congenital, with excess of thin myofilaments 1 ACTA1 Myopathy, congenital, with fiber-type disproportion 1 1 ACTAl Myopathy, nemaline, 3 1 ACTAl Aortic aneurysm, familial thoracic 6 1 ACTA2 Dystonia, juvenile-onset 1 ACTB Cardiomyopathy, dilated, 1R 1 ACTC1 Cardiomyopathy, familial hypertrophic, 11 1 ACTC1 Deafness, autosomal dominant 20/26 1 ACTG 1 Cardiomyopathy, dilated, 1AA 1 ACT 2 Glomerulosclerosis, focal segmental, 1 1 ACTN4 Hereditary hemorrhagic telangiectasia-2 1 ACVRL1 Aminoacylase 1 deficiency 1 ACY1 Alzheimer disease-10 6 AD10 Alzheimer disease-5 6 AD5 Alzheimer disease 6 6 AD6 Alzheimer disease 8 6 AD8 Adenosine deaminase deficiency, partial 1 ADA Thrombotic thrombocytopenic purpura, familial 1 ADAMTS13 ' Adiponectin deficiency 1 ADIPOQ Leukemia, acute myeloid 14 AF10 Mental retardation, X-linked, FRAXE type 1 AFF2 Spinocerebellar ataxia 28 1 AFG3L2 Lipodystrophy, congenital generalized, type 1 1 AGPAT2 Myasthenia, limb-girdle, familial 2 AGRN Hypertension, essential 3 AGTR1 Hyperoxaluria, primary, type 1 1 AGXT Immunodeficiency with hyper-lgM, type 2 1 AICDA Combined oxidative phosphorylation deficiency 6 1 AIFM1 Pituitary adenoma, ACTH-secreting 1 AIP Pituitary adenoma, growth hormone-secreting 1 AIP Pituitary adenoma, prolactin-secreting 1 AIP

Autoimmune polyendocrinopathy syndrome , type 1, with or without reversible metaphyseal dysplasia 1 AIRE Long QT syndrome-11 1 AKAP9 Bile acid synthesis defect, congenital, 2 1 AKR1D1 Breast cancer, somatic 4 AKT1 Colorectal cancer, somatic 6 AKT1 Ovarian cancer, somatic 2 AKT1

Table IX-21781338.1 Diabetes mellitus type II 1 AKT2 Porphyria, acute hepatic 1 ALAD Anemia, sideroblastic, X-linked 1 ALAS2 Protoporphyria, erythropoietic, X-linked dominant 1 ALAS2 Epilepsy, pyridoxine-dependent 1 ALDH7A1 Fructose intolerance 1 ALDOB Congenital disorder of glycosylation, type Ik 1 ALG1 Congenital disorder of glycosylation, type Ig ' 1 ALG12 Congenital disorder of glycosylation, type li 1 ALG2 Congenital disorder of glycosylation, type Id 1 ALG3 Congenital disorder of glycosylation, type lc 1 ALG6 Congenital disorder of glycosylation, type Ih 1 ALG8

Congenital disorder of glycosylation, type II 1 ALG9 Ichthyosiform erythroderma, congenital, nonbullous, 1 2 ALOX12B Ichthyosiform erythroderma, congenital, nonbullous, 1 2 ALOXE3 Hypophosphatasia, adult 1 ALPL Hypophosphatasia, childhood 1 ALPL Hypophosphatasia, infantile 1 ALPL Amyotrophic lateral sclerosis, juvenile 1 ALS2 Primary lateral sclerosis, juvenile 1 ALS2 Spastic paralysis, infantile onset ascending 1 ALS2 Frontorhiny 1 ALX3 Bile acid synthesis defect, congenital, 4 1 AMAC Amelogenesis imperfecta, hypoplastic/hypomaturation type 1 AMELX Leukemia, acute myelogenous 3 AMLCR2 Megaloblastic anemia-1, Norwegian type 1 AMN Glycine encephalopathy 3 AMT Spherocytosis, type 1 1 ANK1 Cardiac arrhythmia, ankyrin-B-related 1 ANK2 Long QT syndrome-4 1 ANK2 Chondrocalcinosis 2 1 ANKH Gnathodiaphyseal dysplasia 1 AN05 Muscular dystrophy, limb-girdle, type 2L 1 AN05 Fibromatosis, juvenile hyaline 1 ANTXR2 Hyalinosis, infantile systemic 1 ANTXR2 Mental retardation, X-linked 59 1 AP1S2 Cerebral palsy, spastic quadriplegic, 3 1 AP4M1 Adenomatous polyposis coli 1 APC Colorectal cancer, somatic 6 APC Gastric cancer, somatic 8 APC Amyloidosis, 3 or more types 1 APOA1

Table IX-21781338.1 Hypoalphalipoproteinemia 1 APOA1 Hypercholesterolemia, familial, modification of 1 APOA2 Hyperchylomicronemia, late-onset 1 APOA5 Hypercholesterolemia, due to ligand-defective apo B 1 APOB Hyperlipoproteinemia, type lb 1 APOC2 Alzheimer disease-2 1 APOE Alzheimer disease 1, familial 6 APP Cerebral amyloid angiopathy, Dutch, Italian, Iowa, Flemish, Arctic variants 1 APP Ataxia, early-onset, with oculomotor apraxia and hypoalbuminemia 1 APTX Coenzyme Q10 deficiency 6 APTX Diabetes insipidus, nephrogenic 1 AQP2 Androgen insensitivity 1 A Androgen insensitivity, partial, with or without breast cancer 1 A R Hypospadias 1, X-linked 1 A R Spinal and bul bar muscular atrophy of Kennedy 1 A R Leukemia, juvenile myelomonocytic 3 ARHGAP26 Slowed nerve conduction velocity, AD 1 ARHGEF10 Leukemia, acute myeloid 14 ARHGEF12 Mental retardation, X-linked nonspecific, type 46 1 ARHGEF6 Maroteaux-Lamy syndrome, several forms 1 ARSB Chondrodysplasia punctata, X-linked recessive 1 ARSE Epilepsy, myoclonic, with mental retardation and spasticity 1 A X Epileptic encephalopathy, early infantile, 1 1 A X Hydranencephaly with abnormal genitalia 1 ARX Lissencephaly, X-linked 2 1 ARX Mental retardation, X-linked 36/43/54 1 ARX Proud syndrome 1 ARX Farber lipogranulomatosis 1 ASAHI Central hypoventilation syndrome, congenital 5 ASCL1 Skin/hair/eye pigmentation 9, dark/light hair 1 ASIP Canavan disease 1 ASPA Microcephaly, primary autosomal recessive, 5, with or without simplified gyral pattern 1 ASPM Alveolar soft part sarcoma 1 ASPSCR1 Citrullinemia , . 1 ASS1 Antithrombin III deficiency 1 AT3 Ataxia, cerebellar, Cayman type 1 ATCAY Parkinson disease 9 1 ATP13A2 Migraine, familial basilar 1 ATP1A2 Migraine, familial hemiplegic, 2 1 ATP1A2

Table IX-21781338.1 Dystonia-12 1 AT 1A3 Hailey-Hailey disease 1 ATP2C1 Mental retardation, X-linked, with epilepsy 1 ATP6AP2 Renal tubular acidosis with deafness 1 ATP6B1 Cutis laxa, autosomal recessive, type II 1 ATP6V0A2 Renal tubular acidosis, distal, autosomal recessive 1 ATP6V0A4 Menkes disease 1 ATP7A Spinal muscular atrophy, distal, X-linked 3 1 ATP7A Cholestasis, benign recurrent intrahepatic 1 ATP8B1 Cholestasis, progressive familial intrahepatic 1 1 ATP8B1 ATP synthase deficiency, nuclear-encoded 1 ATPAF2 Alpha-thalassemia mental retardation syndrome 1 ATRX Alpha-thalassemia myelodysplasia syndrome, somatic 1 ATRX Mental retardation-hypotonic facies syndrome, X-linked 1 ATRX Machado-Joseph disease 1 ATXN3 Spinocerebellar ataxia 8 1 ATXN8 Spinocerebellar ataxia 8 1 ATXN80S Diabetes insipidus, neurohypophyseal 1 AVP Diabetes insipidus, nephrogenic 1 AVPR2 Atrioventricular canal defect 2 AVSD1 Caudal duplication anomaly 1 AXIN1 Hepatocellular carcinoma, somatic 3 AXIN 1 Colorectal cancer . 6 AXIN2 Hypoproteinemia, hypercatabolic 1 B2M Congenital disorder of glycosylation, type lid 1 B4GALT1 Hypercholanemia, familial 3 BAAT Myopathy, myofibrillar, BAG3-related 1 BAG3 Maple syrup urine disease, type la 1 BCKDHA Maple syrup urine disease, type lb 1 BCKDHB Hypercarotenemia and vitamin A deficiency, autosomal dominant 1 BCMOl Leukemia, acute lymphocytic 1 BCR Leukemia, chronic myeloid 1 BCR Mitochondrial complex III deficiency 1 BCS1L Central hypoventilation syndrome, congenital 5 BDNF Spinocerebellar ataxia 31 1 BEAN Microcornea, rod-cone dystrophy, cataract, and posterior staphyloma 1 BEST1 Vitelliform macular dystrophy, adult-onset 1 BEST1 Vitreoretinochoroidopathy. 1 BEST1 Cataract, cortical, juvenile-onset 1 BFSP1 Cataract, autosomal dominant, multiple types 1 1 BFSP2 Cataract, congenital 1 BFSP2

Table IX-21781338.1 Cataract, juvenile-onset 1 BFSP2 Myopathy, centronuclear, autosomal recessive 1 BIN 1 Lymphoproliferative syndrome, X-linked, 2 1 BIRC4 Maturity-onset diabetes of the young, type 11 1 BLK Agammaglobulinemia 4 1 BLNK Ovarian dysgenesis 2 1 BMP15 Premature ovarian failure 4 1 BMP15 Microphthalmia, syndromic 6 1 BMP4 Orofacial cleft 11 1 BMP4 Polyposis syndrome, hereditary mixed, 2 1 BMPR1A Polyposis, juvenile intestinal 2 BMPR1A Chrondrodysplasia, acromesomelic, with genital anomalies 1 BMPR1B Pulmonary hypertension, familial primary 1 BMPR2 Adenocarcinoma of lung, somatic 3 BRAF Fanconi anemia, complementation group Dl 1 BRCA2 Pancreatic cancer 2 BRCA2 Prostate cancer 1 BRCA2 Wilms tumor 1 BRCA2 ' Breast cancer, early-onset 1 BRI P1 Fanconi anemia, complementation group J 1 BRI P1 Mental retardation, X-linked 93 1 BRWD3 Lipodystrophy, congenital generalized, type 2 1 BSCL2 Neuropathy, distal hereditary motor, type V 1 BSCL2 Silver spastic paraplegia syndrome 1 BSCL2 Bartter syndrome, type 4a 1 BSND Sensorineural deafness with mild renal dysfunction 1 BSND Agammaglobulinemia and isolated hormone deficiency 1 BTK Agammaglobulinemia, type 1, X-iinked 1 BTK Colorectal cancer 6 BUB1B Mosaic variegated aneuploidy syndrome 1 BUB1B Mitochondrial DNA depletion syndrome, hepatocerebral form 1 C10orf2 Progressive external ophthalmoplegia with mitochondrial DNA deletions 3 1 C10orf2 Tn syndrome 1 C1GALT1C1 Angioedema, hereditary, types 1and II 1 C1NH Complement component 4, partial deficiency of 1 C1NH Retinal degeneration, late-onset, autosomal dominant 1 C1QTNF5 Brown-Vialetto-Van Laere syndrome 1 C20orf54 Mitochondrial complex 1 deficiency 1 C20orf7 Homocystinuria, cbID type, variant 1 1 C2orf25 Methylmalonic aciduria, cbI D type, variant 2 1 C2orf25

Table IX-21781338.1 C5 deficiency 1 C5 C6 deficiency 1 C6 C7 deficiency . 1 C7 Trichothiodystrophy, nonphotosensitive 1 1 C7orfll Cerebral cavernous malformations-2 1 C7orf22 Osteopetrosis, autosomal recessive 3, with renal tubular acidosis 1 CA2 Cerebellar ataxia and mental retardation with or without quadrupedal locomotion 3 1 CA8 Coenzyme Q10 deficiency 6 CABC1 Night blindness, congenital stationary, type 2B 1 CABP4 Episodic ataxia, type 2 1 CACNA1A Brugada syndrome 3 1 CACNA1C Night blindness, congenital stationary, X-linked, type 2A 1 CACNA1F Retinal cone dystrophy 4 1 CACNA2D4 Brugada syndrome 4 1 CACNB2 Desbuquois dysplasia 1 CANT1 Muscular dystrophy, limb-girdle, type 2A 1 CAPN3 Candidiasis, familial chronic mucocutaneous, autosomal recessive 1 CARD9 FG syndrome 4 1 CASK Mental retardation and microcephaly with pontine and cerebellar hypoplasia 1 CASK Gastric cancer, somatic . 8 CASP10 Non-Hodgkin lymphoma, somatic 1 CASP10 Hepatocellular carcinoma, somatic 3 CASP8 Ventricular tachycardia, catecholaminergic polymorphic, 2 1 CASQ2 Hyperparathyroidism, neonatal 1 CA S Hypocalcemia, autosomal dominant 1 CA S Hypocalciuric hypercalcemia, type 1 1 CASR Male infertility, nonsyndromic, autosomal recessive 1 CATSPER1 Lipodystrophy, congenital generalized, type 3 1 CAV1 Cardiomyopathy, familial hypertrophic 2 CAV3 Creatine phosphokinase, elevated serum 1 CAV3 Long QT syndrome-9 1 CAV3 Muscular dystrophy, limb-girdle, type IC 1 CAV3 Corticosteroid-binding globulin deficiency 1 CBG Homocystinuria, B6-responsive and nonresponsive types 1 CBS Thrombosis, hyperhomocysteinemic 1 CBS 46XY gonadal dysgenesis, complete, CBS2-related 1 CBX2 Mental retardation, autosomal recessive 3 1 CC2D1A COACH syndrome 3 CC2D2A Deafness, autosomal dominant 44 1 CCDC50

Table IX-21781338.1 Thyroid papillary carcinoma 1 CCDC6 Cavernous malformations of CNS and retina 1 CCM1 Cerebral cavernous malformations-1 1 CCM1 Hyperkeratotic cutaneous capillary-venous malformations associated with cerebral capillary malformations 1 CCM1 von Hippel-Lindau disease, modification of 1 CCND1 Neuropathy, hereditary sensory, with spastic paraplegia 1 CCT5 Nephropathy with pretibial epidermolysis bullosa and deafness 1 CD151 Immunodeficiency, common variable, 3 1 CD19 Immunodeficiency due to defect in CD3-zeta 1 CD247 Glomerulosclerosis, focal segmental, 3 1 CD2AP Methylmalonic aciduria due to transcobalamin receptor defect 1 CD320 Platelet glycoprotein IV deficiency 1 CD36 Severe combined immunodeficiency, T cell-negative, B-cell/natural killer-cell positive 3 CD3D Severe combined immunodeficiency, T cell-negative, B-cell/natural killer-cell positive 3 CD3E CD59 deficiency 1 CD59 Immunodeficiency, hypogammaglobulinemia, and reduced B cells 1 CD79B Immunodeficiency, common variable, 6 1 CD81 CD8 deficiency, familial 1 CD8A C-like syndrome 1 CD96 Anemia, congenital dyserythropoietic, type 1 1 CDAN1 Gastric cancer, familial diffuse 1 CDH1 Mental retardation, autosomal dominant 1 CDH15 Deafness, autosomal recessive 12 1 CDH23 Ectodermal dysplasia, ectrodactyly, and macular dystrophy 1 CDH3 Melanoma 1 CDK4 Microcephaly, primary autosomal recessive, 3 1 CDK5RAP2 Angelman syndrome-like 1 CDKL5 Epileptic encephalopathy, early infantile, 2 1 CDKL5 Beckwith-Wiedemann syndrome 1 CDKN1C Pancreatic cancer/melanoma syndrome 1 CDKN2A Leukemia, acute myeloid 14 CEBPA Specific granule deficiency 1 CEBPE Maturity-onset diabetes of the young, type VII I 1 CEL Microcephaly, primary autosomal recessive, 6 1 CEMPJ Hyperalphalipoproteinemia 1 CETP Heterotaxy, visceral, 2, autosomal 1 CFC1 Complement factor 1deficiency 1 CFI Nemaline myopathy 7 1 CFL2 Myasthenic syndrome, congenital, associated with episodic apnea 1 CHAT 8

Table IX-21781338.1 Hypogonadotropic hypogonadism 6 CHD7 Dementia, familial, nonspecific 1 CHMP2B Cataract, posterior polar, 3 1 CHMP4B Multiple pterygium syndrome, lethal type 3 CHRNA1 Myasthenic syndrome, fast-channel congenital 3 CH RNA1 Myasthenic syndrome, slow-channel congenital 4 CH RNA1 Epilepsy, nocturnal frontal lobe, type 4 1 CH RNA2 Epilepsy, nocturnal frontal lobe, 1 1 CHRNA4 Myasthenic syndrome, congenital, associated with acetylcholine receptor deficiency 4 CHRNB1 Myasthenic syndrome, slow-channel congenital 4 CHRNB1 Epilepsy, nocturnal frontal lobe, 3 1 CHRNB2 Multiple pterygium syndrome, lethal type 3 CHRND Myasthenic syndrome, fast-channel congenital 3 CHRND Myasthenic syndrome, slow-channel congenital 4 CHRND Myasthenic syndrome, congenital, associated with acetylcholine receptor deficiency 4 CHRNE Myasthenic syndrome, fast-channel congenital 3 CHRNE Myasthenic syndrome, slow-channel congenital 4 CH RNE Escobar syndrome 1 CHRNG Multiple pterygium syndrome, lethal type 3 CHRNG Chediak-Higashi syndrome 1 CHS1 Spondyloepiphyseal dysplasia with congenital joint dislocations 1 CHST3 Cocoon syndrome 1 CHUK Microphthalmia, isolated 2 1 CHX10 Microphthalmia, isolated, with coloboma 3 1 CHX10 Cirrhosis, North American Indian childhood type 1 CIRH1A Wolfram syndrome 2 1 CISD2 Myotonia congenita, dominant 1 CLCN l Myotonia congenita, recessive 1 CLCN 1 Hypophosphatemic rickets 1 CLCN5 Nephrolithiasis, type 1 1 CLCN5 Proteinuria, low molecular weight, with hypercalciuric nephrocalcinosis 1 CLCN5 Osteopetrosis, autosomal dominant 2 1 CLCN7 , Osteopetrosis, autosomal recessive 4 1 CLCN7 Bartter syndrome, type 4b, digenic 1 CLCNKA Bartter syndrome, type 3 1 CLCNKB Bartter syndrome, type 4, digenic 1 CLCNKB Ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis 1 CLDN1 Hypomagnesemia, primary 1 CLDN16 Hypomagnesemia, renal, with ocular involvement 1 CLDN19

Table IX-21781338.1 Candidiasis, familial chronic mucocutaneous, autosomal dominant 1 CLEC7A Ceroid lipofuscinosis, neuronal 3, juvenile 1 CLN3 Ceroid-lipofuscinosis, neuronal-5, variant late infantile 1 CLN5 Ceroid-lipofuscinosis, neuronal-6, variant late infantile 1 CLN6 Ceroid lipofuscinosis, neuronal 8 1 CLN8 Ceroid lipofuscinosis, neuronal, 8, Northern epilepsy variant 1 CLN8 Macular degeneration juvenile 1 CNGB3 Myopathy, congenital, Compton-North 1 CNTN1 Pitt-Hopkins like syndrome 1 1 CNTNAP2 Deafness, autosomal dominant 9 1 COCH Cerebrocostomandibular-like syndrome 1 COG1 Congenital disorder of glycosylation, type llg 1 COGl Congenital disorder of glycosylation, type llj 1 COG4 Congenital disorder of glycosylation, type e 1 COG7 Congenital disorder of glycosylation, type llh . 1 COG8 Deafness, autosomal dominant 13 1 COLHA2 Deafness, autosomal recessive 53 1 COL11A2 Epidermolysis bullosa, junctional, non-Herlitz type 4 COL17A1 Knobloch syndrome, type 1 1 COL18A1 Caffey disease 1 COL1A1 0 1type II 1 COL1A1 0 1type III 1 COL1A1 0 type IV 1 COL1A1 Epiphyseal dysplasia, multiple, with myopia and deafness 1 COL2A1 Osteoarthritis with mild chondrodysplasia 1 COL2A1 SED congenita 1 COL2A1 , SMED, Strudwick type 1 COL2A1 Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps 1 COL4A1 Brain small vessel disease with Axenfeld-Rieger anomaly 1 COL4A1 Brain small vessel disease with hemorrhage 1 COL4A1 Porencephaly 1 COL4A1 Alport syndrome, autosomal recessive 1 COL4A3 Hematuria, benign familial 1 COL4A3 Alport syndrome, autosomal recessive 1 COL4A4 Alport syndrome 1 COL4A5 Leiomyomatosis, diffuse, with Alport syndrome 1 COL4A6 Ullrich congenital muscular dystrophy 3 COL6A1 Ullrich congenital muscular dystrophy 3 COL6A2 Ullrich congenital muscular dystrophy 3 COL6A3 EBD inversa 1 COL7A1

Table IX-21781338.1 EBD, Bart type 1 COL7A1 Epidermolysis bullosa dystrophica, AD 1 COL7A1 Epidermolysis bullosa dystrophica, A R 1 COL7A1 Epidermolysis bullosa pruriginosa 1 COL7A1 Epidermolysis bullosa, pretibial 1 COL7A1 Toenail dystrophy, isolated 1 COL7A1 Corneal dystrophy polymorphous posterior, 2 1 COL8A2 Corneal dystrophy, Fuchs endothelial, 1 1 COL8A2 Epiphyseal dysplasia, multiple, 2 1 COL9A2 Epiphyseal dysplasia, multiple, 3 1 COL9A3 Endplate acetylcholinesterase deficiency 1 COLQ Epiphyseal dysplasia, multiple 1 1 COMP Coenzyme Q10 deficiency 6 COQ2 Coenzyme Q10 deficiency 6 COQ9 Exocrine pancreatic insufficiency, dyserythropoietic anemia, and calvarial hyperostosis 1 COX4I2 Cytochrome C oxidase deficiency 1 COX6B1 Cerebellar ataxia 1 CP Hemosiderosis, systemic, due to aceruloplasminemia 1 CP Carboxypeptidase N deficiency 1 CPN1 Coproporphyria 1 CPOX Harderoporphyria 1 CPOX Carbamoyl phosphate synthetase 1deficiency 1 CPS1 CPT deficiency, hepatic, type IA 1 CPT1A CPT deficiency, hepatic, type II 1 CPT2 CPT II deficiency, lethal neonatal 1 CPT2 Myopathy due to CPT II deficiency 1 CPT2 Pigmented paravenous chorioretinal atrophy 1 CRB1 Mental retardation, autosomal recessive 2A 1 CRBN Histiocytoma, angiomatoid fibrous, somatic 1 CREB1 Atrioventricular septal defect, partial, with heterotaxy syndrome 2 CRELD1 Inflammatory bowel disease 25 1 CRFB4 Myopathy, cardioskeletal, desmin-related, with cataract 1 CRYAB Cataract, congenital zonular, with sutural opacities 1 CRYBA1 Cataract, lamellar 2 1 CRYBA4 Microphthalmia, isolated, with cataract 4 1 CRYBA4 Cataract, congenital nuclear, autosomal recessive 3 1 CRYBB1 Cataract, cerulean, type 2 1 CRYBB2 Cataract, sutural, with punctate and cerulean opacities 1 CRYBB2 Cataract, congenital nuclear, 2 1 CRYBB3 Cataract, congenital, cerulean type, 3 1 CRYGD

Table IX-21781338.1 Cataract, crystalline aculeiform 1 C YGD Cataract, nonnuclear polymorphic congenital 1 CRYGD Pulmonary alveolar proteinosis 1 CSF2RA Neutrophilia, hereditary 11 CSF3R Chondrosarcoma, extraskeletal myxoid 3 CSMF Night blindness, congenital stationary, type 1 1 CSN B1 Cornelia de Lange syndrome 3 1 CSPG6 Cardiomyopathy, dilated, 1M 1 CSRP3 Cardiomyopathy, familial hypertrophic, 12 1 CSRP3 Cerebral amyloid angiopathy 1 CST3 Macular degeneration, age-related, 11 1 CST3 Epilepsy, progressive myoclonic 1 1 CSTB Hepatocellular carcinoma 2 CTNN B1 Ovarian cancer 1 CTNN B1 Mental retardation in cri-du-chat syndrome 1 CTNN D2 Cystinosis, late-onset juvenile o r adolescent nephropathic 1 CTNS Cystinosis, nephropathic 1 CTNS Cystinosis, ocular nonnephropathic 1 CTNS Periodontitis, juvenile 1 CTSC Ceroid lipofuscinosis, neuronal, 10 1 CTSD Megaloblastic anemia-1, Finnish type 1 CUBN Mental retardation syndrome, X-linked, Cabezas type 1 CUL4B Mental retardation-hypotonic facies syndrome, X-linked, 2 1 CUL4B Methemoglobinemia, type IV 1 CYB5A Methemoglobinemia, type 1 1 CYB5R3 Methemoglobinemia, type II 1 CYB5R3 Chronic granulomatous disease, autosomal, due t o deficiency of CYBA 1 CYBA Chronic granulomatous disease, X-linked 1 CYBB Thrombocytopenia 4 1 CYCS Brooke-Spiegler syndrome 1 CYLD1 Cylindromatosis, familial 1 CYLD1 Trichoepithelioma, multiple familial, 1 1 CYLD1 Lipoid congenital adrenal hyperplasia 1 CYP11A Adrenal hyperplasia, congenital, due t o 11-beta-hydroxylase deficiency 1 CYP11B1 Aldosteronism, glucocorticoid-remediable 1 CYP11B1 Hypoaldosteronism, congenital, due t o CMO 1deficiency 1 CYP11B2 Hypoaldosteronism, congenital, due t o CMO II deficiency 1 CYP11B2 17,20-lyase deficiency, isolated 2 CYP17A1 17-alpha-hydroxylase/17,20-lyase deficiency 2 CYP17A1 Glaucoma 3A, primary congenital 1 CYP1B1 Glaucoma, primary open angle, adult-onset 1 CYP1B1

Table IX-21781338.1 Glaucoma, primary open angle, juvenile-onset 1 CYP1B1 Vitamin D-dependent rickets, type 1 1 CYP27B1 Coumarin resistance 1 CYP2A6 Clopidogrel, impaired responsiveness to 1 CYP2C Mephenytoin poor metabolizer 1 CYP2C Opremazole poor metabolizer 1 CYP2C Proguanil poor metabolizer 1 CYP2C Warfarin sensitivity 1 CYP2C9 Rickets due to defect in vitamin D 25-hydroxylation 1 CYP2R1 Ichthyosis, lamellar, 3 1 CYP4F22 Spastic paraplegia-5A 1 CYP7B1 D-2-hydroxyglutaric aciduria 1 D2HGDH Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism 1 DAX1 Dosage-sensitive sex reversal 1 DAX1 Maple syrup urine disease, type II 1 DBT Mirror movements, congenital 1 DCC Severe combined immunodeficiency, Athabascan type 1 DCLRE1C Corneal dystrophy, congenital stromal 1 DCN Neuropathy, distal hereditary motor, type VIIB 1 DCTN1 Spondylometaepiphyseal dysplasia, short limb-hand type 1 DDR2 Cardiomyopathy, dilated, 1 1 1 DES Myopathy, desmin-related, cardioskeletal 1 DES Deafness, autosomal dominant 5 1 DFNA5 Hyperoxaluria, primary, type III 1 DHDPSL Miller syndrome 1 DHODH Pseudohyperkalemia, familial 1 DHS Deafness, autosomal dominant 1 1 DIAPH1 Premature ovarian failure 1 DIAPH2 Mental retardation, FRA12A type 1 DIP2B Renal cell carcinoma 2 DIRC2 Schizophrenia 1 DISC2 Parkinson disease 7, autosomal recessive early-onset 1 DJ1 Maple syrup urine disease, type III 1 DLD Esophageal cancer 1 DLEC1 Lung cancer 5 DLEC1 Spondylocostal dysostosis, autosomal recessive, 1 1 DLL3 Amelogenesis imperfecta, hypomaturation-hypoplastic type, with taurodontism 1 DLX3 Cardiomyopathy, dilated, 3B 1 DMD Hypophosphatemic rickets, A R 1 DMP1 Myotonic dystrophy 1 DMPK

Table IX-21781338.1 Ciliary dyskinesia, primary, 7, with or without situs inversus 1 DNAH11 Ciliary dyskinesia, primary, 3, with or without situs inversus 1 DNAH5 Ciliary dyskinesia, primary, 1, with or without situs inversus 1 DNAI1 Ciliary dyskinesia, primary, 9, with or without situs inversus 1 DNAI2 3-Methylglutaconic aciduria type V 1 DNAJC19 Charcot-Marie-Tooth disease, axonal, type 2M 1 DNM2 Charcot-Marie-Tooth disease, dominant intermediate B 1 DNM2 Myopathy, centronuclear 2 DNM2 Immunodeficiency-centromeric instability-facial anomalies syndrome 1 DNMT3B Hyper-lgE recurrent infection syndrome, autosomal recessive 1 DOCK8 Myasthenia, limb-girdle, familial 2 DOK7 Congenital disorder of glycosylation, type Ij 1 DPAGT2 Congenital disorder of glycosylation, type le 1 DPMI Congenital disorder of glycosylation, type lo 1 DPM3 Ventricular fibrillation, paroxysmal familial, 2 1 DPP6 5-fluorouracil toxicity 1 DPYD Dystonia, myoclonic 1 D D2 Arrhythmogenic right ventricular dysplasia, familial, 11 1 DSC2 Hypotrichosis and recurrent skin vesicles 1 DSC3 Arrhythmogenic right ventricular dysplasia, familial, 10 1 DSG2 Cardiomyopathy, dilated, IBB 1 DSG2 Hypotrichosis, localized, autosomal recessive 1 DSG4 Arrhythmogenic right ventricular dysplasia 8 1 DSP Dilated cardiomyopathy with woolly hair and keratoderma 1 DSP Epidermolysis bullosa, lethal acantholytic 1 DSP Deafness, autosomal dominant 36, with dentinogenesis 1 DSPP Thryoid dyshormonogenesis 6 1 DUOX2 Thyroid dyshormonogenesis 5 1 DUOXA2 Cornelia de Lange syndrome 2 1 DXS423E Asphyxiating thoracic dystrophy 3 1 DYNC2H1 Muscular dystrophy, limb-girdle, type 2B 1 DYSF Myopathy, distal, with anterior tibial onset 1 DYSF Dystonia-1, torsion 1 DYTl Chondrodysplasia punctata, X-linked dominant 1 EBP Ectodermal dysplasia, anhidrotic, X-linked 1 EDI Tooth agenesis, selective, X-linked 1 1 EDI Ectodermal dysplasia, hypohidrotic, autosomal dominant 1 EDAR Ectodermal dysplasia, hypohidrotic, autosomal recessive 1 EDAR Ectodermal dysplasia, anhidrotic, autosomal dominant 1 EDARADD Ectodermal dysplasia, anhidrotic, autosomal recessive 1 EDARADD Central hypoventilation syndrome, congenital 5 EDN3

Table IX-21781338.1 Migraine, resistance t o 1 EDNRA ABCD syndrome 1 EDNRB Doyne honeycomb degeneration of retina 1 EFEMP1 Cutis laxa, autosomal recessive, type 1 1 EFEMP2 Hypomagnesemia 4, renal 1 EGF Adenocarcinoma of lung, response t o tyrosine kinase inhibitor in 1 EGFR Nonsmall cell lung cancer, response t o tyrosine kinase inhibitor in 1 EGFR Erythrocytosis, familial, 3 1 EGLN1 Neuropathy, congenital hypomyelinating, 1 1 EGR2 Leukoencephalopathy with vanishing white matter 4 EIF2B1

Leukoencephalopathy with vanishing white matter 4 EI F2B2 Leukoencephalopathy with vanishing white matter 4 EIF2B3

Leukoencephaly with vanishing white matter 1 EI F2B4 Leukoencephalopathy with vanishing white matter 4 EIF2B5 Hematopoiesis, cyclic 1 ELANE Neutropenia, severe congenital, autosomal dominant 1 1 ELAN E Cutis laxa, AD 1 ELN Macular dystrophy, autosomal dominant, chromosome 6-linked 1 ELOVL4 Emery-Dreifuss muscular dystrophy 1 EMD Bowen-Conradi syndrome . 1 EMG1 Amelogenesis imperfecta, type IB 1 ENAM Amelogenesis imperfecta, type IC 1 ENAM Hereditary hemorrhagic telangiectasia-1 1 ENG Arterial calcification, generalized, of infancy 1 ENPP1 Hypophosphatemia rickets, autosomal recessive, 2 1 ENPP1 Ossification of posterior longitudinal ligament of spine 1 ENPP1 Colorectal cancer 6 EP300 Erythrocytosis, familial, 4 1 EPAS1 Elliptocytosis-1 1 EPB41 Spherocytosis, hereditary, type 5 1 EPB42 Stomatocytosis 1 1 EPB72 Colorectal cancer, hereditary nonpolyposis, type 1 1 EPCAM Diarrhea 5, with tufting enteropathy, congenital 1 EPCAM Cataract, posterior polar, 1 1 EPHA2 Prostate cancer, progression and metastasis of 1 EPHB2 Hypercholanemia, familial 3 EPHX1 Epilepsy, myoclonic, Lafora type 2 EPM2A Eosinophil peroxidase deficiency 1 EPX Adenocarcinoma of lung, somatic 3 ERBB2 Gastric cancer, somatic 8 ERBB2 Glioblastoma, somatic 1 ERBB2

Table IX-21781338.1 Cerebrooculofacioskeletal syndrome 4 1 ERCC1 Cerebrooculofacioskeletal syndrome 2 1 ERCC2 Trichothiodystrophy 1 ERCC2 Trichothiodystrophy 1 ERCC3 XFE progeroid syndrome 1 ERCC4 Cerebrooculofacioskeletal syndrome 1 1 ERCC6 Cockayne syndrome type B 1 ERCC6 De Sanctis-Cacchione syndrome 1 ERCC6 Cockayne syndrome type A 1 ERCC8 Deafness, autosomal recessive 36 1 ESPN Deafness, autosomal recessive 35 1 ESRRB Glutaricaciduria, type IIA 1 ETFA

Glutaricaciduria, type MB 1 ETFB Glutaricaciduria, type IIC 1 ETFDH Leukemia, acute myeloid, somatic 1 ETV6 Neuroepithelioma 1 EWSR1 Exostoses, multiple, type 1 1 EXT1 Exostoses, multiple, type 2 1 EXT2 Branchiootorenal syndrome with cataract 1 EYA1

Cardiomyopathy, dilated, 1J 1 EYA4 Deafness, autosomal dominant 10 1 EYA4 Factor XI deficiency, autosomal dominant 1 Fll Factor XI deficiency, autosomal recessive 1 Fll Angioedema, hereditary, type III 1 F12 Factor XII deficiency 1 F12 Factor XIIIA deficiency 1 F13A1 Factor XIIIB deficiency 1 F13B Factor V deficiency 1 F5 Hemophilia B 1 F9 Thrombophilia, X-linked, due t o factor IX defect 1 F9 Leukodystrophy, dysmyelinating, and spastic paraparesis with o r without dystonia 1 FA2H Fanconi anemia, complementation group B 1 FAAP95 Osteopathia striata with cranial sclerosis 1 FAM123B Leukodystrophy, hypomyelinating, 5 1 FAM126A Neuropathy, hereditary sensory and autonomic, type IIB 1 FAM134B STAR syndrome 1 FAM58A Amelogenesis imperfecta, type 3 1 FAM83H Fanconi anemia, complementation group A 1 FANCA Fanconi anemia, complementation group 1 1 FANCI Mitochondrial complex IV deficiency 1 FASTKD2

Table IX-21781338.1 Cutis laxa, autosomal dominant 1 FBLN5 Cutis laxa, autosomal recessive 1 FBLN5 Macular degeneration, age-related, 3 1 FBLN5 MASS syndrome 2 FBN1 Contractural arachnodactyly, congenital 1 FBN2 Parkinson disease 15, autosomal recessive 1 FBX07 Thrombocytopenic purpura, autoimmune 1 FCGR2C Polycystic kidney and hepatic disease 1 FCYT Protoporphyria, erythropoietic, autosomal dominant 1 FECH Protoporphyria, erythropoietic, autosomal recessive 1 FECH Afibrinogenemia, congenital 2 FGA Amyloidosis, hereditary renal 1 FGA Afibrinogenemia, congenital 2 FGB Aplasia of lacrimal and salivary glands 1 FGF10 LADD syndrome 2 FGF10 Hypophosphatemia rickets, autosomal dominant 1 FGF23 Tumoral calcinosis, hyperphosphatemic, familial 1 FGF23 Deafness, congenital with inner ear agenesis, microtia, and microdontia 1 FGF3 Hypogonadotropic hypogonadism 6 FGFR1 Jackson-Weiss syndrome 2 FGFR1 Trigonocephaly 1 FGFR1 Beare-Stevenson cutis gyrata syndrome 1 FGFR2 Gastric cancer, somatic 8 FGFR2 . Saethre-Chotzen syndrome 2 FGFR2 Bladder cancer, somatic 3 FGFR3 CATSHL syndrome 1 FGFR3 Cervical cancer, somatic 1 FGFR3 Colorectal cancer, somatic 6 FGFR3 LADD syndrome 2 FGFR3 Nevus, keratinocytic, nonepidermolytic 1 FGFR3 Leiomyomatosis and renal cell cancer 1 FH Multiple cutaneous and uterine leiomyomata 1 FH Emery-Dreifuss muscular dystrophy 6 1 FHL1 Myopathy, reducing body, X-linked, childhood-onset 1 FHL1 Myopathy, reducing body, X-linked, severe early-onset 1 FHL1 Myopathy, X-linked, with postural muscle atrophy 1 FHL1 Premature ovarian failure 6 1 FIGLA Cardiomyopathy, dilated, I X 1 FKTN Colorectal cancer, somatic 6 FLCN Pneumothorax, primary spontaneous 1 FLCN Renal carcinoma, chromophobe, somatic 1 FLCN

Table IX-21781338.1 Ichthyosis vulgaris 1 FLG Thrombocytopenia-2 1 FU 14813 Premature ovarian failure 2B 1 FU22792 FG syndrome 2 1 FLNA Heterotopia, periventricular 1 FLNA Heterotopia, periventricular, ED variant 1 FLNA Intestinal pseudoobstruction, neuronal 1 FLNA Melnick-Needles syndrome 1 FLNA Larsen syndrome . 1 FLNB Spondylocarpotarsal synostosis syndrome 1 FLNB Myopathy, myofibrillar, filamin C-related 1 FLNC Leukemia, acute myeloid 14 FLT3 Hemangioma, capillary infantile, somatic 2 FLT4 Lymphedema, hereditary 1 1 FLT4 Proliferative vasculopathy and hydraencephaly-hydrocephaly syndrome 1 FLVCR2 Glomerulopathy with fibronectin deposits 2 1 FN1 Neurodegeneration due t o cerebral folate transport deficiency 1 FOLR1 Iridogoniodysgenesis, type 1 1 FOXC1 Iris hypoplasia and glaucoma 1 FOXC1 Rieger or Axenfeld anomalies 1 FOXC1 Lymphedema-distichiasis syndrome 1 FOXC2 Lymphedema-distichiasis syndrome with renal disease and diabetes mellitus 1 FOXC2 Anterior segment mesenchymal dysgenesis 2 FOXE3 Aphakia, congenital primary 1 FOXE3 Alveolar capillary dysplasia with misalignment of pulmonary veins 1 FOXF1 Rett syndrome, congenital variant 1 FOXG1B Enlarged vestibular aqueduct 2 FOXI 1 Premature ovarian failure 3 1 FO L2 T-cell immunodeficiency, congenital alopecia, and nail dystrophy 1 FOXN1 Rhabdomyosarcoma, alveolar 1 FOXOIA Speech-language disorder-1 1 FOXP2 Immunodysregulation, polyendocrinopathy, and enteropathy, X-linked 1 FOXP3 Bifid nose with or without anorectal and renal anomalies 1 FREM1 Nystagmus 1, congenital, X-linked 2 FRMD7 Follicle-stimulating hormone deficiency, isolated 1 FSHB Ovarian dysgenesis 1 1 FSHR Ovarian response t o FSH stimulation 1 FSHR Polycystic ovary syndrome 1 FST Glutamate formiminotransferase deficiency 1 FTCD Hyperferritinemia-cataract syndrome 1 FTL

Table IX-21781338.1 Neurodegeneration with brain iron accumulation 3 1 FTL Growth retardation, developmental delay, coarse facies, and early death 1 FTO Mental retardation, X-linked-9 1 FTSJ 1 Hypomagnesemia-2, renal 1 FXYD2 Exudative vitreoretinopathy 1 FZD4 Retinopathy of prematurity 1 FZD4 Darsun syndrome 1 G6PC3 Neutropenia, severe congenital, autosomal recessive 4 1 G6PC3 Epilepsy, juvenile myoclonic, susceptibility t o 1 GABRD Epilepsy, generalized, with febrile seizures plus, type 3 1 GABRG2 Febrilel, convulsions, familial 1 GABRG2 Myoclonic epilepsy, severe, of infancy 1 GABRG2 Cerebral palsy, spastic, symmetric, autosomal recessive 1 GAD1 Krabbe disease 1 GALC Galactose epimerase deficiency 1 GALE Tumoral calcinosis, hyperphosphatemic, familial 1 GALNT3 Galactosemia 1 GALT GAMT deficiency 1 GAMT Charcot-Marie-Tooth disease type 2D 1 GARS Neuropathy, distal hereditary motor, type V 1 GARS Dyserythropoietic anemia with thrombocytopenia 1 GATA1 Leukemia, megakaryoblastic, of Down syndrome 1 GATA1 Leukemia, megakaryoblastic, with o r without Down syndrome 1 GATA1 Macrothrombocytopenia 1 GATA1 Thrombocytopenia with beta-thalassemia, X-linked 1 GATA1 Hypoparathyroidism, sensorineural deafness, and renal dysplasia 1 GATA3 AGAT deficiency 1 GATM Glutaricaciduria, type 1 1 GCDH Dystonia, DOPA-responsive, with or without hyperphenylalainemia 1 GCH1 Hyperpehnylalaninemia, BH4-deficient, B 1 GCH 1 Diabetes mellitus, gestational 1 GCK Diabetes mellitus, noninsulin-dependent, late onset 1 GCK Diabetes mellitus, permanent neonatal 1 GCK Hyperinsulinemic hypoglycemia, familial, 3 1 GCK Hemolytic anemia due t o gamma-glutamylcysteine synthetase deficiency 1 GCLC Hypoparathyroidism, familial isolated 1 GCM B Adult i phenotype with congenital cataract 1 GCNT2 Adult i phenotype without cataract 1 GCNT2 Congenital disorder of glycosylation, type lib 1 GCS1

Table IX-21781338.1 Glycine encephalopathy 1 GCSH Charcot-Marie-Tooth disease, axonal, type 2K 1 GDAP1 Charcot-Marie-Tooth disease, axonal, with vocal cord paresis 1 GDAP1 Charcot-Marie-Tooth disease, recessive intermediate, A 1 GDAP1 Chondrodysplasia, Grebe type 1 GDF5 Symphalangism, proximal 2 GDF5 Klippel-Feil syndrome, autosomal dominant 1 GDF6 Microphthalmia, isolated 4 1 GDF6 Spondylocostal dystostosis 4, autosomal dominant 1 GDF6 Mental retardation, X-linked nonspecific 1 GDI1 Central hypoventilation syndrome 1 GDNF Myopathy, mitochondrial progressive, with congenital cataract, hearing loss, and developmental delay 1 G FE Neutropenia, nonimmune chronic idiopathic, of adults 1 GFI1 Neutropenia, severe congenital, autosomal dominant 2 1 GFI1 Combined oxidative phosphorylation deficiency 1 1 GFM1 Pseudoxanthoma elasticum-like disorder with multiple coagulation factor deficiency 1 GGCX Vitamin K-dependent coagulation defect 1 GGCX Growth hormone deficiency, isolated, type IA 1 GH1 Growth hormone deficiency, isolated, type IB 1 GH1 Growth hormone deficiency, isolated, type II . 1 GH1 Kowarski syndrome 1 GH1 Short stature, idiopathic 1 GHR Growth hormone deficiency, isolated, type IB 1 GHRH R Short stature 1 GHSR Intrinsic factor deficiency 1 GIF Parkinson disease 11 1 GIGYF2 Fibromatosis, gingival, 2 1 GINGF2 Atrioventricular septal defect 2 GJA1 Hallermann-Streiff syndrome 1 GJA1 Hypoplastic left heart syndrome 1 GJA1 Cataract, zonular pulverulent-3 1 GJA3 Atrial fibrillation 1 GJA5 Cataract, zonular pulverulent-1 1 GJA8 Cataract-microcornea syndrome 1 GJA8 Charcot-Marie-Tooth neuropathy, X-linked dominant, 1 1 GJB1 Bart-Pumphrey syndrome 1 GJB2 Deafness, autosomal dominant 3A 1 GJB2 Deafness, autosomal recessive 1A 1 GJB2 Hystrix-like ichthyosis with deafness 1 GJB2

Table IX-21781338.1 Keratitis-ichthyosis-deafness syndrome 1 GJB2 Vohwinkel syndrome 1 GJB2 Deafness, autosomal dominant 2B 1 GJB3 Deafness, digenic, GJB2/GJB3 1 GJB3 Deafness, autosomal dominant 3 B 1· GJB6 Deafness, autosomal recessive B 1 GJB6 Deafness, digenic GJB2/GJB6 1 GJB6 Ectodermal dysplasia, hidrotic 1 GJB6 Leukodystrophy, hypomyelinating, 2 . 1 GJC2 Lymphedema, hereditary, IC 1 GJC2 Spastic paraplegia, 44 1 GJC2 GMl-gangliosidosis, type 1 1 GLB1 Morquio syndrome B 1 GLB1 Glaucoma IB, primary open angle, adult onset 1 GLC1B Glycine encephalopathy 3 GLDC Arthrogryposis, lethal, with anterior horn cell disease 1 GLE1 Holoprosencephaly-9 1 GLI2 Polydactyly, postaxial, types Al and B 1 GLI3 Polydactyly, preaxial, type IV 1 GLI3 Nephronophthisis 7 1 GLIS2 Diabetes mellitus, neonatal, with congenital hypothyroidism 1 GLIS3 Startle disease/hyperekplexia, autosomal dominant 1 GLRA1 Hyperekplexia, autosomal recessive 1 GLRB Anemia, sideroblastic, pyridoxine-refractory, autosomal recessive 1 GLRX5 Glutamine deficiency, congenital 1 GLUL Leukemia, acute myelogenous 3 GMPS Ventricular tachycardia, idiopathic 1 GNAI2 Osseous heteroplasia, progressive 1 GNAS Night blindness, congenital stationary, autosomal dominant 3 1 GNAT1 Inclusion body myopathy, autosomal recessive 1 GNE Nonaka Myopathy 1 GNE Glycine N-methyltransferase deficiency 1 GNMT Chondrodysplasia punctata, rhizomelic, type 2 1 G PAT Hypogonadotropic hypogonadism due t o GNRH deficiency 1 GNRH1 Fertile eunuch syndrome 1 GNRHR Mucopolysaccharidosis type I D 1 GNS Thyroid carcinoma, papillary 6 GOLGA5 Globozoospermia 2 GOPC Wilms tumor, somatic 1 GPC3 Omodysplasia 1 1 GPC6 Brugada syndrome 2 1 GPD1L

Table IX-21781338.1 Hyperekplexia 2 GPHN Molybdenum cofactor deficiency, type C 1 GPHN Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency 1 GPI Nystagmus 6, congenital, X-linked 2 GPR143

Ocular albinism, type 1, Nettleship-Falls type 1 GPR143 Polymicrogyria, bilateral frontoparietal 1 GPR56 Convulsions, familial febrile, 4 1 GPR98 Deafness, autosomal dominant 28 1 GRHL2 Hyperoxaluria, primary, type II 1 GRHPR Mental retardation, X-linked 94 1 GRIA3 Mental retardation, autosomal recessive, 6 1 GRIK2 Night blindness, congenital stationary, type I B 1 GRM6 Frontotemporal lobar degeneration with ubiquitin-positive inclusions 1 GRN Deafness, autosomal recessive 25 1 GRXCR1 Amyloidosis, Finnish type 1 GSN Hemolytic anemia due t o glutathione synthetase deficiency 1 GSS Trichothiodystrophy, complementation group A 1 GTF2H5 Cone dystrophy-3 1 GUCA1A Mucopolysaccharidosis VII 1 GUSB Glycogen storage disease 0, muscle 1 GYS1 Silver-Russell syndrome 1 H19 Wilms tumor 2 1 H19 Cortisone reductase deficiency 1 H6PD Fatty liver, acute, of pregnancy 1 HADHA HELLP syndrome, maternal, of pregnancy 1 HADHA LCHAD deficiency 1 HADHA Trifunctional protein deficiency 2 HADHA Trifunctional protein deficiency 2 HADHB 3-hydroxyacyl-coa dehydrogenase deficiency 1 HADHSC Hyperinsulinemic hypoglycemia, familial, 4 1 HADHSC Neutropenia, severe congenital, autosomal recessive 3 1 HAX1 Heinz body anemias, alpha- 1 HBA1 Thalassemias, alpha- 2 HBA1 Thalassemia, alpha- 2 HBA2 Heinz body anemias, beta- 1 HBB Thalassemia-beta, dominant inclusion-body 1 HBB Thalassemias, beta- 1 HBB Fetal hemoglobin quantitative trait locus 1 2 HBG 1 Fetal hemoglobin quantitative trait locus 1 2 HBG2 Microphthalmia, syndromic 7 1 HCCS

Table IX-21781338.1 Thrombophilia due t o heparin cofactor II deficiency 1 HCF2 Brugada syndrome 8 1 HCN4 Growth hormone deficiency with pituitary anomalies 1 HESX1 GM2-gangliosidosis, several forms 1 HEXA Basal laminar drusen 1 HF1 Complement factor H deficiency 1 HF1 Hemolytic uremic syndrome, atypical, susceptibility to, 1 1 HF1 Membranoproliferative glomerulonephritis with CFH deficiency 1 HF1 Deafness, autosomal recessive 39 1 HGF 3-hydroxyisobutryl-CoA hydrolase deficiency 1 HIBCH Hemolytic anemia due to hexokinase deficiency 1 HK1 Porphyria, acute intermittent 1 HMBS Porphyria, acute intermittent, nonerythroid variant 1 HMBS HMG-CoA synthase-2 deficiency 1 HMGCS2 Diabetes mellitus, insulin-dependent, 20 1 HNF1A Hepatic adenoma 1 HNF1A Renal cell carcinoma 2 HNF1A Diabetes mellitus, noninsulin-dependent 1 HNF1B Microtia, hearing impairment, and cleft palate 1 HOXA2 Vertical talus, congenital 1 HOXD10 Brachydactyly type D 1 HOXD13 Brachydactyly-syndactyly syndrome 1 HOXD13 Syndactyly, type V 1 HOXD13 Digital clubbing, isolated congenital 1 HPGD Hypertrophic osteoarthropathy, primary, autosomal recessive 1 HPGD HPRT-related gout 1 H T1 Urofacial syndrome 1 HPSE2 Hypotrichosis, hereditary, Marie Unna type, 1 1 HR Costello syndrome 1 HRAS Thrombophilia due t o elevated HRG 1 HRG Thrombophilia due t o HRG deficiency 1 HRG Hyperparathyroidism, familial primary 1 HRPT2 Hyperparathyroidism-jaw tumor syndrome 1 HRPT2 Parathyroid adenoma with cystic changes 1 HRPT2 Parathyroid carcinoma 1 HRPT2 Cortisone reductase deficiency 1 HSD11B1 17-beta-hydroxysteroid dehydrogenase X deficiency 1 HSD17B10 Mental retardation, X-linked 17/31, microduplication 1 HSD17B10 Mental retardation, X-linked syndromic 10 1 HSD17B10 Pseudohermaphroditism, male, with gynecomastia 1 HSD17B3 D-bifunctional protein deficiency 1 HSD17B4

Table IX-21781338.1 Cholestasis, progressive familial intrahepatic 4 1 HSD3B7 Cataract, lamellar 1 HSF4 Cataract, Marner type 1 HSF4 Charcot-Marie-Tooth disease, axonal, type 2 F 1 HSPB1 Neuropathy, distal hereditary motor, type B 1 HSPB1 Neuronopathy, distal hereditary motor, type IIC 1 HSPB3 Charcot-Marie-Tooth disease, axonal, type 2L 1 HSPB8 Neuropathy, distal hereditary motor, type IIA 1 HSPB8 Leukodystrophy, hypomyelinating, 4 1 HSPD1 Spastic paraplegia-13 1 HSPD1 Parkinson disease 13 1 HT A2 Mental retardation, X-linked syndromic, Turner type 1 HUWE1 Cleft lip/palate-ectod.ermal dysplasia syndrome 1 HVEC Orofacial cleft 7 1 HVEC Mucopolysaccharidosis type IX 1 HYAL1 Hydrolethalus syndrome 1 HYLS1 Ichthyosis, congenital, autosomal recessive 1 ICHYN Immunodeficiency, common variable, 1 1 ICOS BCG infection, generalized familial 1 IFNGR1 Mycobacterial infection, atypical, familial disseminated 2 IFNGR1 Cranioectodermal dysplasia 1 IFT122 Asphyxiating thoracic dystrophy 2 1 IFT80 Corpus callosum, agenesis of, with mental retardation, ocular coloboma and micrognathia 1 IGBP1 Growth retardation with deafness and mental retardation due t o IGF1 deficiency 1 IGF1

Insulin-like growth factor 1, resistance to 1 IGF1R Agammaglobulinemia 1 1 IGHM Neuronopathy, distal hereditary motor, type V I 1 IGHMBP2 Agammaglobulinemia 2 1 IGLL1 Dysautonomia, familial 1 IKBKAP Ectodermal dysplasia, hypohidrotic, with immune.deficiency 1 IKBKG Ectodermal, dysplasia, anhidrotic, lymphedema and immunodeficiency 1 IKBKG Immunodeficiency, isolated 1 IKBKG BCG and salmonella infection, disseminated 1 IL12B Mental retardation, X-linked, 21/34 1 IL1RAPL1 Interleukin 1 receptor antagonist deficiency 1 IL1RN lnterleukin-2 receptor, alpha chain, deficiency of 1 IL2RA Combined immunodeficiency, X-linked, moderate 1 IL2RG Severe combined immunodeficiency, X-linked 1 IL2RG Severe combined immunodeficiency, T-cell negative, B-cell/natural killer cell-positive type. 1 IL7R 24

Table IX-21781338.1 Glomerulosclerosis, focal segmental, 5 1 INF2 Mental retardation, truncal obesity, retinal dystrophy, and micropenis 1 INPP5E Diabetes mellitus, insulin-dependent, 2 1 INS Diabetes mellitus, permanent neonatal 1 INS Diabetes mellitus, type 1 1 INS Maturity-onset diabetes of the young, type 10 1 INS Cryptorchidism, idiopathic 2 INSL3 Diabetes mellitus, insulin-resistant, with acanthosis nigricans 1 INSR . Hyperinsulinemic hypoglycemia, familial, 5 1 INSR Leprechaunism 1 INSR Nephronophthisis 2, infantile 1 INVS Pancreatic agenesis 1 IPF1 Mental retardation, X-linked 1 1 IQSEC2 Invasive pneumococcal disease, recurrent isolated, 1 1 IRAK4 I A K4 deficiency 1 IRAK4 Gastric cancer, somatic 8 IRF1 Nonsmall cell lung cancer, somatic 2 IRF1 Orofacial cleft 6 1 IRF6 Popliteal pterygium syndrome 1 IRF6 Myopathy with lactic acidosis, hereditary 1 ISCU Autoimmune disease, syndromic multisystem 1 ITCH Epidermolysis bullosa, junctional, with pyloric stenosis 1 ITGA6 Muscular dystrophy, congenital, due t o ITGA7 deficiency 1 ITGA7 Epidermolysis bullosa of hands and feet 1 ITGB4 Epidermolysis bullosa, junctional, non-Herlitz type 4 ITGB4 Epidermolysis bullosa, junctional, with pyloric atresia 1 ITGB4 Lymphoproliferative syndrome, EBV-associated, autosomal, 1 1 ITK Dementia, familial British 1 ITM2B Dementia, familial Danish 1 ITM2B Spinocerebellar ataxia 15 1 ITPR1 Thyroid dyshormonogenesis 4 1 IYD Leukemia, acute myelogenous 3 JAK2 Myelofibrosis, idiopathic 1 JAK2 Polycythemia vera 1 JAK2 Thrombocythemia, essential 3 JAK2 Arrhythmogenic right ventricular dysplasia, familial, 12 1 JUP Cerebral palsy, spastic quadriplegic 1 KANK1 Charcot-Marie-Tooth disease, recessive intermediate, B 1 KARS Episodic ataxia/myokymia syndrome 1 KCNA1 Atrial fibrillation, familial, 7 1 KCNA5 ' Atrial fibrillation, familial, 4 1 KCNE2

Table IX-21781338.1 Brugada syndrome 6 1 KCNE3 Short QT syndrome-1 1 KCNH2 Bartter syndrome, type 2 1 KCNJ 1 Diabetes mellitus, permanent neonatal, with neurologic features 1 KCNJ 11 Diabetes mellitus, transient neonatal, 3 1 KCNJ 11 Diabetes, permanent neonatal 1 KCNJ 11 Hyperinsulinemic hypoglycemia, familial, 2 1 KCNJ 11 Long QT syndrome-7 1 KCNJ2 Short QT syndrome-3 1 KCNJ2 Long QT syndrome 13 1 KCNJ5 Birk-Barel mental retardation dysmorphism syndrome 1 KCNK9 Atrial fibrillation, familial, 3 1 KCNQ1 Long QT syndrome-1 1 KCNQ1 Short QT syndrome-2 1 KCNQ1 Epilepsy, benign, neonatal, type 1 1 KCNQ2 Myokymia with neonatal epilepsy 1 KCNQ2 Epilepsy, benign neonatal, type 2 1 KCNQ3 Deafness,. autosomal dominant 2A 1 KCNQ4 Retinal cone dystrophy 3 B 1 KCNV2 Epilepsy, progressive myoclonic 3 1 KCTD7 Mental retardation, X-linked, syndromic, JARIDlC-related 1 KDM5C Hemangioma, capillary infantile, somatic 2 KDR Cornea plana congenita, recessive 1 KERA Spastic paraplegia-8 . 1 KIAA0196 Charcot-Marie-Tooth disease type 2A1 1 KIF1B Pheochromocytoma 5 KIF1B Fibrosis of extraocular muscles, congenital, 1 1 KIF21A Fibrosis of extraocular muscles, congenital, 3 B 1 KIF21A Spastic paraplegia 10 1 KIF5A Kindler syndrome 2 KIND1 Leukocyte adhesion deficiency, type III 1 KIND3 Mental retardation, autosomal dominant 4 1 KIRREL3 Hypogonadotropic hypogonadism 6 KISS1R Germ cell tumors 1 KIT Leukemia, acute myeloid 14 KIT Hyperpigmentation, familial progressive 1 KITLG Tumoral calcinosis, hyperphosphatemic 1 KL Blood group-Lutheran inhibitor 1 KLF1 Maturity-onset diabetes of the young, type VII 1 KLF11 Gastric cancer, somatic 8 KLF6 Prostate cancer, somatic 2 KLF6

Table IX-21781338.1 Hodgkin lymphoma 1 KLHDC8B Amelogenesis imperfecta, type MAI 1 hyperkeratosis 2 KRT1 Ichthyosis, cyclic, with epidermolytic hyperkeratosis 2 KRT1 Plamoplantar keratoderma, epidermolytic 1 KRT1 Epidermolytic hyperkeratosis 2 KRT10 Ichthyosis with confetti 1 KRT10 Ichthyosis, cyclic, with epidermolytic hyperkeratosis 2 KRT10 Nevus, epidermal, epidermolytic hyperkeratotic type 1 KRT10 Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT14 Epidermolysis bullosa simplex, Koebner type 2 KRT14 Epidermolysis bullosa simplex, recessive 1 KRT14 Epidermolysis bullosa simplex, Weber-Cockayne type 2 KRT14 Palmoplantar keratoderma, nonepidermolytic 1 KRT16 Palmoplantar keratoderma, nonepidermolytic, focal 1 KRT16 Palmoplantar verrucous nevus, unilateral 1 KRT16 Steatocystoma multiplex 1 KRT17 Ichthyosis bullosa of Siemens 1 KRT2 Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT5 Epidermolysis bullosa simplex, Koebner type 2 KRT5 Epidermolysis bullosa simplex, Weber-Cockayne type 2 KRT5 Ectodermal dysplasia, 'pure' hair-nail type 1 KRT85 Ciliary dyskinesia, primary, 10 1 KTU Corpus callosum, partial agenesis of 1 L1CAM CRASH syndrome 1 L1CAM Hydrocephalus due to aqueductal stenosis 1 L1CAM Hydrocephalus with congenital idiopathic intestinal pseudoobstruction 1 L1CAM Hydrocephalus with Hirschsprung disease and cleft palate 1 L1CAM Muscular dystrophy, congenital merosin-deficient 1 LAMA2 Muscular dystrophy, congenital, due t o partial LAMA2 deficiency 1 LAMA2 Epidermolysis bullosa, generalized atrophic benign 1 LAMA3 Epidermolysis bullosa, junctional, Herlitz type 3 LAMA3 Laryngoonychocutaneous syndrome 1 LAMA3 Nephrosis, congenital, with or without ocular abnormalities 1 LAMB2 Epidermolysis bullosa, junctional, Herlitz type 3 LAMB3

Table IX-21781338.1 Epidermolysis bullosa, junctional, non-Herlitz type 4 LAMB3 Epidermolysis bullosa, junctional, Herlitz type 3 LAMC2 Epidermolysis bullosa, junctional, non-Herlitz type 4 LAMC2 Arrhythmogenic right ventricular dysplasia 5 1 LAMR1 Ellis-van Creveld syndrome 1 LBN Pelger-Huet anomaly 1 LBR Norum disease 2 LCAT Thalassemia, Hispanic gamma-delta-beta 1 LCRB Lactase deficiency, congenital 1 LCT Cardiomyopathy, dilated 1C 1 LDB3 Myopathy, myofibrillar, ZASP-related 1 LDB3 Hypercholesterolemia, familial 1 LDLR Hypercholesterolemia, familial, autosomal recessive 1 LDLRAP1 Osteopoikilosis 1 LEM D3 Spondylocostal dysostosis, autosomal recessive 3 1 LFNG Epilepsy, partial, with auditory features 1 LGI1 Cryptorchidism, bilateral 2 LGR8 Leydig cell hypoplasia with pseudohermaphroditism 1 LHCGR Luteinizing hormone resistance, female 1 LHCGR Deafness, autosomal recessive 67 1 LH FPL5 Pituitary hormone deficiency, combined, 3 1 LHX3 Pituitary hormone deficiency, combined, 4 1 LHX4

Hypotrichosis, localized, autosomal recessive 2 1 LI PH Acheiropody 1 LM BR1 Polydactyly, preaxial type II 1 LM BR1 Lipase deficiency, combined 1 LMF1 Cardiomyopathy, dilated, 1A 1 LM A Emery-Dreifuss muscular dystrophy, AD 1 LM A Emery-Dreifuss muscular dystrophy, A 1 LMNA Huchinson-Gilford Progeria 1 LMNA Lipodystrophy, familial partial 1 LMNA Muscular dystrophy, limb-girdle, type I B 1 LMNA Leukodystrophy, adult-onset, autosomal dominant 1 LMNB1 Lipodystrophy, partial, acquired 1 LMNB2 Osteosarcoma 3 LOH18CR1 Vohwinkel syndrome with ichthyosis 1 LOR Cutis laxa, recessive, type 1 1 LOX Deafness, autosomal recessive 77 1 LOXHD1 Myoglobinuria, acute recurrent, autosomal recessive 1 LPIN1 Combined hyperlipidemia, familial 1 LPL Lipoprotein lipase deficiency 1 LPL

Table IX-21781338.1 Leukemia, acute myeloid 14 LPP Retinal dystrophy, early-onset severe 1 LRAT Exudative vitreoretinopathy 4 1 LRP5 Hyperostosis, endosteal 1 LRP5 Osteopetrosis, AD type 1 1 LRP5 Osteoporosis-pseudoglioma syndrome 1 LRP5 van Buchem disease, type 2 1 LRP5 Ciliary dyskinesia, primary, 13 1 LRRC50 Agammaglobulinemia 5 1 LRRC8A Parkinson disease-8 1 LRRK2 Deafness, autosomal recessive 63 1 LRTOMT Glaucoma 3,' primary congenital, D 1 LTBP2 Tooth agenesis, selective, 6 1 LTBP3 Amyloidosis, renal 1 LYZ Esophageal squamous cell carcinoma 2 LZTS1 Prostate cancer, somatic 2 MAD1L1 Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome 1 MADH4 Polyposis, juvenile intestinal 2 MADH4 Pulmonary hypertension, primary 1 MADH9 Mental retardation, X-linked 95 1 MAGT1 Hypospadias 2, X-linked 1 MAMLD1 Mannosidosis, alpha-, types 1and II 1 MAN2B1 Manhosidosis, beta 1 MAN BA Brunner syndrome 1 MAOA Lung cancer, somatic 1 MAP3K8 Immunodeficiency due to defect in MAPBP-interacting protein 1 MAPBPIP Epileptic encephalopathy, Lennox-Gastaut type 1 MAPK10 Dementia, frontotemporal, with or without parkinsonism 1 MAPT Pick disease 2 MAPT Supranuclear palsy,- progressive 1 MAPT Supranuclear palsy, progressive atypical 1 MAPT Deafness, autosomal recessive 49 1 MARVELD2 Hypermethioninemia, persistent, autosomal dominant, due to methionine adenosyltransferase l/lll deficiency 1 MAT1A Methionine adenosyltransferase deficiency, autosomal recessive 1 MAT1A Epiphyseal dysplasia, multiple, 5 1 MATN3 Spondyloepimetaphyseal dysplasia 1 MATN3 Myopathy, distal 2 1 MATR3 Mental retardation, autosomal dominant 1 1 MBD5 Glucocorticoid deficiency, due to ACTH unresponsiveness 1 MC2R Obesity, autosomal dominant 1 MC4R

Table IX-21781338.1 3-Methylcrotonyl-CoA carboxylase 1 deficiency 1 MCCC1 3-Methylcrotonyl-CoA carboxylase 2 deficiency 1 MCCC2 Methylmalonyl-CoA epimerase deficiency 1 MCEE Factor V and factor VIII, combined deficiency of 1 MCFD2 Lactase persistance/nonpersistance 1 MCM6 Microcephaly, autosomal recessive 1 1 MCPH1 Premature chromosome condensation with microcephaly and mental retardation 1 MCPH1 Microcephaly, primary autosomal recessive, 2 1 MCPH2 Microcephaly, primary autosomal recessive, 4 1 MCPH4 Angelman syndrome 1 MECP2 Encephalopathy, neonatal severe 1 MECP2 Mental retardation, X-linked, Lubs type 1 MECP2 Mental retardation, X-linked, syndromic 13 1 MECP2 Rett syndrome, preserved speech variant 1 MECP2 Lujan-Fryns syndrome 1 MED12 Opitz-Kaveggia syndrome 1 MED12 Transposition of the great arteries, dextro-looped 1 1 MED13L Chromosome 5ql4.3 deletion syndrome 1 MEF2C Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations 1 MEF2C Hyperparathyroidism, AD 1 MEN 1 Spondylocostal dysostosis, autosomal recessive 2 1 MESP2 Hepatocellular carcinoma, childhood type 1 MET Renal cell carcinoma, papillary, familial and sporadic 1 MET Charcot-Marie-Tooth disease type 2A2 1 MFN2 Microphthalmia, isolated 5 1 MFRP Ceroid lipofuscinosis, neuronal, 7 1 MFSD8 Congenital disorder of glycosylation, type IIA 1 MGAT2 Bare lymphocyte syndrome, type II, complementation group A 1 MHC2TA Opitz G syndrome, type 1 1 MIDI Thyroid carcinoma, follicular 2 MINPP1 Cataract, polymorphic and lamellar 1 MIP Mirror-image Polydactyly 1 MIPOL1 Deafness, autosomal dominant 50 1 MIR96 Leukemia, acute myeloid 14 MLF1 Colorectal cancer, hereditary nonpolyposis, type 2 1 MLH1 Mismatch repair cancer syndrome 1 MLH1 Colorectal cancer, somatic ' 6 MLH3 Endometrial cancer 1 MLH3 Methylmalonic aciduria, vitamin B12-responsive 1 MMAA

Table IX-21781338.1 Methylmalonic aciduria, vitamin B12-responsive due to defect in synthesis of adenosylcobalamin, cblB complementation type 1 MMAB COPD, rate of decline of lung function in 1 MMP1 Amelogenesis imperfecta, type IIA2 1 MMP20 Meningioma 1 MN1 Molybdenum cofactor deficiency, type A 1 MOCS1 Molybdenum cofactor deficiency, type B 1 MOCS2 Congenital disorder of glycosylation, type If 1 MPDU1 Carbohydrate-deficient glycoprotein syndrome, type lb 1 MPI Thrombocythemia, essential 3 MPL Thrombocytopenia, congenital amegakaryocytic 1 MPL Mitochondrial DNA depletion syndrome, hepatocerebral form 1 MPV17 Charcot-Marie-Tooth disease type 2 1 1 MPZ Charcot-Marie-Tooth disease type 2J 1 MPZ Charcot-Marie-Tooth disease, dominant intermediate 3 1 MPZ Neuropathy/congenital hypomyelinating 1 MPZ Paroxysmal nonkinesigenic dyskinesia 1 MR1 Glucocorticoid deficiency 2 1 M AP Combined oxidative phosphorylation deficiency 2 1 MRPS16 Combined oxidative phosphorylation deficiency 5 1 MRPS22 Immunodeficiency, common variable, 5 1 MS4A1 Colorectal cancer, hereditary nonpolyposis, type 1 1 MSH2 Mismatch repair cancer syndrome 1 MSH2 Endometrial cancer, familial 1 MSH6 Mismatch repair cancer syndrome 1 MSH6 Prostate cancer, hereditary 1 MSR1 Orofacial cleft 5 1 MSX1 Tooth agenesis, selective, 1, with or without orofacial cleft 1 MSX1 Witkop syndrome 1 MSX1 Craniosynostosis, type 2 1 MSX2 Myotubular myopathy, X-linked 1 MTM1 Charcot-Marie-Tooth disease type 4B1 1 MTMR2 Abetalipoproteinemia 1 MTP Homocystinuria-megaloblastic anemia, cbl Etype 1 MTRR Myasthenic syndrome, congenital, associated with acetylcholine receptor deficiency 4 MUSK Adenomas, multiple colorectal 1 MUTYH Colorectal adenomatous polyposis, autosomal recessive, with pilomatricomas 1 MUTYH Gastric cancer, somatic 8 MUTYH Hyper-lgD syndrome 1 MVK Cardiomyopathy, familial hypertrophic, 4 1 MYBPC3 31

Table IX-21781338.1 Microcephaly and digital abnormalities with normal intelligence 1 MYCN Myopathy, centronuclear 2 MYF6 Aortic aneurysm, familial thoracic 4 1 MYH11 Deafness, autosomal dominant 4 1 MYH14 Inclusion body myopathy-3 1 MYH2 Arthrogryposis, distal, type 2A 1 MYH3 Arthrogryposis, distal, type 2B 2 MYH3 Cardiomyopathy, dilated, 1EE 1 MYH6 Cardiomyopathy, familial hypertrophic, 14 1 MYH6 Cardiomyopathy, dilated, I S 1 MYH7 Cardiomyopathy, familial hypertrophic, 1 1 MYH7 Myopathy, myosin storage 1 MYH7 Carney complex variant 1 MYH8 Deafness, autosomal dominant 17 1 MYH9 Epstein syndrome 1 MYH9 Fechtner syndrome 1 MYH9 Macrothrombocytopenia and progressive sensorineural deafness 1 MYH9 May-Hegglin anomaly 1 MYH9 Sebastian syndrome 1 MYH9 Cardiomyopathy, familial hypertrophic, 10 1 MYL2 Cardiomyopathy, familial hypertrophic, 8 1 MYL3 Cardiomyopathy, hypertrophic, midventricular, digenic 1 MYLK2 Deafness, autosomal recessive 3 1 MY015A Deafness, autosomal dominant 48 1 MYOIA Deafness, autosomal recessive 30 1 MY03A Deafness, autosomal dominant 22 1 MY06 Deafness, autosomal recessive 37 1 MY06 Deafness, sensorineural, with hypertrophic cardiomyopathy 1 MY06 Deafness, autosomal dominant 11, neurosensory 1 MY07A Deafness, autosomal recessive 2, neurosensory 1 MY07A Glaucoma 1A, primary open angle, juvenile-onset 1 MYOC Kanzaki disease 1 NAGA Schindler disease, type 1 1 NAGA Schindler disease, type II I 1 NAGA N-acetylglutamate synthase deficiency 1 NAGS Familial cold autoinflammatory syndrome 2 1 NALP12 Hydatidiform mole 1 NALP7 Chronic granulomatous disease due to deficiency of NCF-1 1 NCF1 Chronic granulomatous disease due to deficiency of NCF-2 1 NCF2 Thyroid carcinoma, papil lary 6 COA4 Exudative vitreoretinopathy, X-linked 1 NDP

Table IX-21781338.1 Mitochondrial complex 1deficiency 1 NDUFA1 Mitochondrial complex 1deficiency 1 NDUFA11 Mitochondrial complex 1deficiency 1 NDUFAF2 Mitochondrial complex 1deficiency 1 NDUFAF3 Mitochondrial complex 1deficiency 1 NDU FAF4 Mitochondrial complex 1deficiency 1 NDU FS1 Mitochondrial complex 1deficiency 1 NDU FS2 Mitochondrial complex 1deficiency 1 NDUFS4

Complex 1, mitochondrial respiratory chain, deficiency of 1 NDUFS6 Alexander disease 1 NDUFV1 Mitochondrial complex 1deficiency 1 NDUFV1 Mitochondrial complex 1deficiency 1 NDUFV2 Nemaline myopathy 2, autosomal recessive 1 NEB Charcot-Marie-Tooth disease type 2 E 1 NEFL Hypogonadotropic hypogonadism 6 NELF Maturity-onset diabetes of the young 6 1 NEU OD1 Diarrhea 4, malabsorptive, congenital 1 NEUROG3 Cardiomyopathy, dilated, ICC 1 NEXN Leukemia, juvenile myelomonocytic 3 NF1 Neurofibromatosis, familial spinal 1 NF1 Neurofibromatosis-Noonan syndrome 1 NF1 Meningioma, NF2-related, somatic 1 NF2 Schwannomatosis 1 NF2 Ectodermal dysplasia, anhidrotic, with T-cell immunodeficiency 1 NFKBIA Neuropathy, hereditary sensory and autonomic, type V 1 NGFB Severe combined immunodeficiency with microcephaly, growth retardation, and sensitivity to ionizing radiation 1 NHEJ1 Epilepsy, myoclonic, Lafora type 2 NHLRC1 Epilepsy, progressive myoclonic 2 B 1 NHLRC1 Cataract, congenital, X-linked 1 NHS Spastic paraplegia-6 1 NIPA1 Chorea, hereditary benign 3 NKX2-1 Choreoathetosis, hypothyroidism, and neonatal respiratory distress 1 NKX2-1 Persistent truncus arteriosus 1 NKX2-6 Atrial septal defect with atrioventricular conduction defects 1 NKX2E Hypothyroidism, congenital nongoitrous, 5 1 NKX2E Mental retardation, X-linked 1 LGN4 Cold-induced autoinflammatory syndrome, familial 1 NLRP3 Neuroblastoma 7 NME1 Premature ovarian failure 5 1 NOBOX Heterotaxy, visceral, 5 1 NODAL

Table IX-21781338.1 Symphalangism, proximal 2 NOG Synostoses syndrome, multiple, 1 1 NOG Alzheimer disease, late-onset, susceptibility to 6 NOS3 Aortic valve disease 1 NOTCH1 Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy 1 NOTCH3 Nephronophthisis 1, juvenile 1 NPHP1 Nephronophthisis 3 1 NPHP3 Nephronophthisis 4 1 NPHP4 Nephrotic syndrome, type 1 1 NPHS1 Leukemia, acute myeloid 14 NPM1 Atrial fibrillation, familial, 6 1 NPPA Obesity, mild, early-onset 1 NR0B2 Enhanced S-cone syndrome 1 NR2E3 Hypertension, early-onset, autosomal dominant, with exacerbation in pregnancy 1 NR3C2 Premature ovarian failure 7 1 NR5A1 Anemia, hypochromic microcytic 1 NRAMP2 Colorectal cancer 6 NRAS Thyroid carcinoma, follicular 2 NRAS Leukemia, acute myeloid 14 NSD1 Anemia, hemolytic, due to UMPH1 deficiency 1 NT5C3 Glaucoma 1, open angle, 10 1 NTF4 Insensitivity to pain, congenital, with anhidrosis 1 NTRK1 Medullary thyroid carcinoma, familial 1 NTRK1 Leukemia, acute myeloid 14 NUP214 Striatonigral degeneration, infantile 1 NUP62 Three M syndrome 2 1 OBSL1 Albinism, brown oculocutaneous 2 OCA2 Albinism, oculocutaneous, type II 2 OCA2 Lowe syndrome 1 OCRL Alpha-ketoglutarate dehydrogenase deficiency 1 OGDH Renal cell carcinoma, clear cell, somatic 1 OGG1 Optic atrophy 1 1 OPA1 Optic atrophy and deafness 1 OPA1 3-Methylglutaconic aciduria type III 1 OPA3 Optic atrophy and cataract 1 OPA3 Mental retardation, X-linked, with cerebellar hypoplasia and distinctive facial appearance 1 OPHN 1 Blue-cone monochromacy 1 OPN1LW Colorblindness, deutan 1 OPN1MW Colorblindness, tritan 1 OPN1SW 34

Table IX-21781338.1 Amytrophic lateral sclerosis 12 1 OPTN Glaucoma 1, open angle, E 1 OPTN Immune dysfunction with T-cell inactivation due t o calcium entry defect 1 1 ORAI1 Amyloidosis, primary localized cutaneous 1 OSM R Osteopetrosis, autosomal recessive 5 1 OSTM1 Deafness, autosomal recessive 22 1 OTOA Auditory neuropathy, autosomal recessive, 1 1 OTOF Deafness, autosomal recessive 9 1 OTOF Microphthalmia, syndromic 5 1 OTX2 Bleeding disorder due t o P2 Y12 defect 1 P2RY12 Hypotrichosis, localized, autosomal recessive, 3 1 P2RY5 Lissencephaly-1 1 PAFAH1B1 Phenylketonuria 1 PAH Plasminogen activator inhibitor, type 1 1 PAI1 Mental retardation, X-linked 30 1 PAK3 Fanconi anemia, complementation group N 1 PALB2 HARP syndrome 1 PANK2 Neurodegeneration with brain iron accumulation 1 1 PANK2 SEMD, Pakistani type 1 PAPSS2 Optic nerve coloboma with renal disease 1 PAX2 Rhabdomyosarcoma 2, alveolar 2 PAX3 Diabetes mellitus, ketosis-prone 1 PAX4 Diabetes mellitus, type 2 1 PAX4 Maturity-onset diabetes of the young, type IX 1 PAX4 Aniridia 1 PAX6 Cataract with late-onset corneal dystrophy 1 PAX6 Coloboma of optic nerve 1 PAX6 Coloboma, ocular 2 PAX6 Gillespie syndrome 1 PAX6 Keratitis 1 PAX6 Optic nerve hypoplasia 1 PAX6 Rhabdomyosarcoma 2, alveolar 2 PAX7 Hypothyroidism, congenital, due t o thyroid dysgenesis or hypoplasia 1 PAX8 Tooth agenesis, selective, 3 1 PAX9 Hyperphenylalaninemia, BH4-deficient, D 1 PCBD Deafness, autosomal recessive 23 1 PCDH15 Epilepsy, female-restricted, with mental retardation 1 PCDH19 Thyroid carcinoma, papillary 1 PCM1 Obesity with impaired prohormone processing 1 PCSK1 Hypercholesterolemia, familial, 3 1 PCSK9

Table IX-21781338.1 Paget disease of bone 3 PDB4 Cerebral cavernous malformations 3 1 PDCD10 Nephrotic syndrome, type 2 1 PDCN Pigmented nodular adrenocortical disease, primary, 2 1 PDE11A Night blindness, congenital stationary, autosomal dominant 2 1 PDE6B Cone dystrophy 4 1 PDE6C Retinal cone dystrophy 3 1 PDE6H Striatal degeneration, autosomal dominant 1 PDE8B Hypereosinophilic syndrome, idiopathic, resistant t o imatinib 1 PDGFRA Myeloproliferative disorder with eosinophilia 1 PDGFRB Colorectal cancer 6 PDGFRL Hepatocellular cancer 1 PDGFRL Leigh syndrome, X-linked 1 PDHA1 Pyruvate dehydrogenase deficiency 1 PDHA1 Coenzyme Q10 deficiency 6 PDSS1 Coenzyme Q10 deficiency 6 PDSS2 Lacticacidemia due t o PDX1 deficiency 1 PDX1 Advanced sleep phase syndrome, familial 1 PER2 Adrenoleukodystrophy, neonatal 5 PEX1 Refsum disease, infantile 1 PEX1 Adrenoleukodystrophy, neonatal 5 PEX10 Adrenoleukodystrophy, neonatal 5 PEX13 Adrenoleukodystrophy, neonatal 5 PEX26 Refsum disease, infantile form 2 PEX26 Adrenoleukodystrophy, neonatal 5 PEX5 Properdin deficiency, X-linked 1 PFC Phosphoglycerate kinase 1 deficiency 1 PGK1 Spastic paraplegia-7 1 PGN Progesterone resistance 1 PGR Hypophosphatemia, X-linked 1 PHEX Mental retardation syndrome, X-linked, Siderius type 1 PHF8 Phosphoglycerate dehydrogenase deficiency 1 PHGDH Muscle glycogenosis 1 PHKA1 Phosphorylase kinase deficiency of liver and muscle, autosomal recessive 1 PHKB Fibrosis of extraocular muscles, congenital, 2 1 PHOX2A Leukemia, acute myeloid 14 PICALM Glycosylphosphatidylinositol deficiency 1 PIGM Breast cancer, somatic 4 PIK3CA Colorectal cancer, somatic 6 PIK3CA Gastric cancer, somatic 8 PIK3CA

Table IX-21781338.1 Hepatocellular carcinoma, somatic 3 PIK3CA Nevus, epidermal 1 PIK3CA Nonsmall cell lung cancer, somatic 2 PIK3CA Ovarian cancer, somatic 2 PIK3CA Corneal fleck dystrophy 1 PIKFYVE Parkinson disease 6, early onset 1 PINK1 Clubfoot, congenital 1 PITX1 Iridogoniodysgenesis, type 2 1 PITX2 Anterior segment mesenchymal dysgenesis 2 PITX3 Cataract, posterior polar, 4 1 PITX3 Cataract, posterior polar, 4, syndromic 1 PITX3 Deafness, autosomal recessive 59 1 PJVK Polycystic kidney disease, adult type 1 1 PKD1 Polycystic kidney disease 2 1 PKD2 Adenosine triphosphate, elevated, of erythrocytes 1 PKLR Pyruvate kinase deficiency 1 PKLR Ectodermal dysplasia/skin fragility syndrome 1 PKP1 Arrhythmogenic right ventricular dysplasia, familial, 9 1 PKP2 Dystonia-parkinsonism, adult-onset 1 PLA2G6 Karak syndrome 1 PLA2G6 Neurodegeneration with brain iron accumulation 2B 1 PLA2G6 Adenomas, salivary gland pleomorphic 1 PLAG1 Hyperfibrinolysis, familial, due t o increased release of PLAT 1 PLAT Thrombophilia, familial, due t o decreased release of PLAT 1 PLAT Nephrotic syndrome, type 3 1 PLCE1 Muscular dystrophy with epidermolysis bullosa simplex 1 PLEC1 Spinal muscular atrophy, distal, autosomal recessive, 4 1 PLEKHG5 Osteopetrosis, autosomal recessive 6 1 PLEKHM1 Conjunctivitis, ligneous 1 PLG Alpha-2-plasmin inhibitor deficiency 1 PLI Cardiomyopathy, dilated, I P 1 PLN Nevo syndrome 1 PLOD Lysyl hydroxylase 3 deficiency 1 PLOD3 Spastic paraplegia-2 1 PLP1 Congenital disorder of glycosylation, type la 1 PMM2 Neuropathy, recurrent, with pressure palsies 1 PMP22 Mismatch repair cancer syndrome 1 PMS2 Central hypoventilation syndrome, congenital 5 PMX2B Hirschsprung disease, short-segment 1 PMX2B Microcephaly, seizures, and developmental delaty 1 PNKP Hypertension, essential 3 PNMT

Table IX-21781338.1 Immunodeficiency due t o purine nucleoside phosphorylase deficiency 1 PNP Spastic paraplegia 39 1 PNPLA6 N syndrome 1 POLA Progressive external ophthalmoplegia, autosomal dominant, with or without hypogonadism 1 POLG Progressive external ophthalmoplegia, autosomal recessive 1 POLG Progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant 4 1 POLG 2 Obesity, adrenal insufficiency, and red hair due t o POMC deficiency 1 POMC Adrenal hyperplasia, congenital, due t o combined P450C17 and P450C21 deficiency 1 POR Antley-Bixler syndrome-like with disordered steroidogenesis 1 POR Disordered steroidogenesis, isolated 1 POR POR deficiency 1 POR Pituitary hormone deficiency, combined, 1 1 POU1F1 Deafness, X-linked 2 1 POU3F4 Deafness, autosomal dominant 15 1 POU4F3 Carotid intimal medial thickness 1 1 PPARG Insulin resistance, severe, digenic 1 PPARG Lipodystrophy, familial partial, type 3 1 PPARG Obesity, severe 2 PPARG Breast cancer 3 PPM1D Porphyria variegata 1 PPOX Insulin resistance, severe, digenic 1 PPP1R3A Lung cancer 5 PPP2R1B Spinocerebellar ataxia 12 1 PPP2R2B Ceroid lipofuscinosis, neuronal 1, infantile 1 PPT1 Renpenning syndrome 1 PQBP1 Renal cell carcinoma, papillary, 1 2 PRCC Hemophagocytic lymphohistiocytosis, familial, 2 1 PRF1 Lymphoma, non-Hodgkin 1 PRF1 Epilepsy, progressive myoclonic I B 1 PRICKLEl Cardiomyopathy, hypertrophic 6, with WPW 1 PRKAG2 Wolff-Parkinson-White syndrome 1 PRKAG2 Myxoma, intracardiac 1 PRKAR1A Pigmented adrenocortical disease, primary, 1 1 PRKAR1A Thyroid carcinoma, papillary 6 PRKAR1A Spinocerebellar ataxia 14 1 PRKCG Polycystic liver disease 2 PRKCSH Adenocarcinoma of lung, somatic 3 PRKN Adenocarcinoma, ovarian, somatic 1 PRKN Parkinson disease, juvenile, type 2 1 PRKN 38

Table IX-21781338.1 Creutzfeldt-Jakob disease 1 PRNP Gerstmann-Straussler disease 1 PRNP Insomnia, fatal familial 1 PRNP Prion disease with protracted course 1 PRNP Thrombophilia due t o protein C deficiency, autosomal dominant 1 PROC Thrombophilia due t o protein C deficiency, autosomal recessive 1 PROC Hypogonadism, hypogonadotropic 1 PROK2 Macular dystrophy, retinal, 2 1 PROM1 Pituitary hormone deficiency, combined, 2 1 PROP1 Thrombophilia due t o protein S deficiency 1 PROS1 Choriodal dystrophy, central areolar 2, 1 PRPH2 Foveomacular dystrophy, adult-onset, with choroidal neovascularization 1 PRPH2 Macular dystrophy, patterned ' 1 PRPH2 Macular dystrophy, vitelliform 1 PRPH2 Arts syndrome 1 PRPS1 Charcot-Marie-Tooth disease, X-linked recessive, 5 1 PRPS1 Deafness, X-linked 1 1 PRPS1 Gout, PRPS-related 1 PRPS1 Pancreatitis, hereditary 1 PRSS1 Mental retardation, autosomal recessive 1 1 PRSS12 Enterokinase deficiency 1 PRSS7 Combined SAP deficiency 1 PSAP Gaucher disease, atypical 1 PSAP Krabbe disease, atypical 1 PSAP Alzheimer disease, type 3 3 PSEN 1 Alzheimer disease, type 3, with spastic paraparesis and apraxia 3 PSEN 1 Alzheimer disease, type 3, with spastic paraparesis and unusual plaques 3 PSEN 1 Dementia, frontotemporal 1 PSEN 1 Pick disease 2 PSEN 1 Alzheimer disease-4 1 PSEN2 Pyogenic sterile arthritis, pyoderma gangrenosum, and acne 1 PSTPIP1 Basal cell carcinoma, somatic 3 PTC HI Basal cell nevus syndrome 1 PTCH1 Holoprosencephaly-7 1 PTCH1 Basal cell carcinoma, somatic 3 PTCH2 Medulloblastoma 1 PTCH2 Cowden disease 1 PTEN VATER association with macrocephaly and ventriculomegaly 1 PTEN Diabetes mellitus, permanent neonatal, with cerebellar agenesis 1 PTF1A Hypertension, essential 3 PTGIS Hypoparathyroidism, autosomal dominant 1 PTH

Table IX-21781338.1 Hypoparathyroidism, autosomal recessive 1 PTH Failure of tooth eruption, primary 1 PTHR1 Leopard syndrome 1 PTPN 11 Leukemia, juvenile myelomonocytic 3 PTPN11 Severe combined immunodeficiency, T cell-negative, B-cell/natural killer-cell positive 3 PTPRC Colon cancer, somatic 1 PTPRJ Deafness, autosomal recessive 84 1 PTPRQ Lipodystrophy, congenital generalized, type 4 1 PTRF Hyperphenylalaninemia, BH4-deficient, A 1 PTS Ectodermal dysplasia-syndactyly syndrome 1 1 PVRL4 efsum disease, infantile form 2 PXMP3 Cutis laxa, autosomal recessive, type MB 1 PYCR1 Mental retardation, joint hypermobility and skin laxity, with o r without metabolic abnormalities 1 PYCS McArdle disease 1 PYGM Hyperphenylalaninemia, BH4-deficient, C 1 QDPR Mental retardation, X-linked-72 1 RAB39B Warburg micro syndrome 1 1 RAB3GAP1 Martsolf syndrome 1 RAB3GAP2 Charcot-Marie-Tooth disease type 2B 1 RAB7 Fanconi anemia, complementation group 0 1 RAD51C Breast cancer, invasive ductal 1 RAD54L Lymphoma, non-Hodgkin, somatic 1 RAD54L LEOPARD syndrome 2 1 RAF1 Alpha/beta T-cell lymphopenia with gamma/delta T-cell expansion, severe cytomegalovirus infection, and autoimmunity 1 RAG1 Combined cellular and humoral immune defects with granulomas 2 RAG1 Severe combined immunodeficiency, B cell-negative 2 RAG1 Combined cellular and humoral immune defects with granulomas 2 RAG2 Severe combined immunodeficiency, B cell-negative 2 RAG2 Myasthenic syndrome, congenital, associated with acetylcholine receptor deficiency 4 RAPSN Myasthenic syndrome, congenital, associated with facial dysmorphism and acetylcholine receptor deficiency 1 RAPSN Leukemia, acute promyelocytic 1 RARA Basal cell carcinoma, somatic 3 RASA1 Capillary malformation-arteriovenous malformation 1 RASA1 Lung cancer 5 RASSF1 Microphthalmia, isolated 3 1 RAX Bladder cancer 3 RBI Osteosarcoma 3 RBI

Table IX-21781338.1 Breast cancer, somatic 4 RB1CC1 Cardiomyopathy, dilated, 1DD 1 RBM20 Alopecia, neurologic defects, and endocrinopathy syndrome 1 RBM28 Deafness, autosomal recessive, 24 1 RDX Bloom syndrome 1 RECQL3 Spastic paraplegia 31 1 REEP1 Hyperuricemic nephropathy, familial juvenile 2 1 REN Central hypoventilation syndrome, congenital 5 RET Medullary thyroid carcinoma 1 RET Multiple endocrine neoplasia IIA 1 RET Multiple endocrine neoplasia IIB 1 RET Pheochromocytoma 5 RET Renal agenesis 1 RET Congenital disorder of glycosylation, type In 1 RFT1 Bare lymphocyte syndrome, type II, complementation group C 1 RFX5 Bare lymphocyte syndrome, type II, complementation group E 1 RFX5 MHC class II deficiency, complementation group B 1 RFXAN K Bare lymphocyte syndrome, type II, complementation group D 1 RFXAP Anemia, hemolytic, Rh-null, regulator type 1 RHAG Night blindness, congenital stationery, autosomal dominant 1 1 RHO Macrocephaly, alopecia, cutis laxa, and scoliosis 1 RIN2 Newfoundland. rod-cone dystrophy 1 RLBP1 Rippling muscle disease-1 1 RMD1 Anauxetic dysplasia 1 RMRP Metaphyseal dysplasia without hypotrichosis 1 RMRP Prostate cancer 1, 176807 1 RNASEL Leukoencephalopathy, cystic, without megalencephaly 1 RNASET2 Renal cell carcinoma 2 RNF139 RIDDLE syndrome 1 RNF168 Recombination rate QTL 1 1 RN F212 Esophageal carcinoma, somatic 1 RN F6 Vesicoureteral reflux 2 1 ROB02 Gaze palsy, horizontal, with progressive scoliosis 1 ROB03 Robinow syndrome, autosomal recessive 1 ROR2 . COACH syndrome 3 RPGRIP1L Ribose-5-phosphate isomerase deficiency 1 RPIA Diamond-Blackfan anemia 7 1 RPL11 Diamond-Blackfan anemia 5 1 RPL35A Diamond-Blackfan anemia 6 1 RPL5 Diamond-Blackfan anemia 9 1 RPS10 Macrocytic anemia, refractory, due t o 5q deletion, somatic 1 RPS14

4 1

Table IX-21781338.1 Diamond-Blackfan anemia 4 1 RPS17 Diamond-Blackfan anemia 1 1 RPS19 Diamond-blackfan anemia 1 RP524 Diamond-Blackfan anemia 10 1 RPS26 Diamond-Blackfan anemia 8 1 RPS7 Mitochondrial DNA depletion syndrome, encephalomyopathic form, with renal tubulopathy 1 RRM2B Progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant, 5 1 RRM2B Ciliary dyskinesia, primary, 11 1 RSPH4A Ciliary dyskinesia, primary, 12 1 RSPH9 Palmoplantar hyperkeratosis and true hermaphroditism 1 RSPOl Anonychia congenita 1 RSP04 Leukemia, acute myeloid 14 RUNX1 Platelet disorder, familial, with associated myeloid malignancy 1 RUNX1 Minicore myopathy with external ophthalmoplegia 1 RYR1 Neuromuscular disease, congenital, with uniform type 1 fiber 1 RYR1 Arrhythmogenic right ventricular dysplasia 2 1 RYR2 Ventricular tachycardia, catecholaminergic polymorphic, 1 1 RYR2 Spastic ataxia, Charlevoix-Saguenay type 1 SACS Tumoral calcinosis, familial, normophosphatemic 1 SAMD9 Porokeratosis, disseminated superficial actinic, 1 1 SART3 Cleft palate and mental retardation 1 SATB2 Epilepsy, generalized, with febrile seizures plus, type 2 1 SCN 1A Epilepsy, severe myoclonic, of infancy 1 SCN 1A Febrile convulsions, familial, 3A 1 SCN1A Migraine, familial hemiplegic, 3 1 SCN 1A Brugada syndrome 5 1 SCN 1B Cardiac conduction defect, nonspecific 1 SCN 1B Generalized epilepsy with febrile seizures plus 1 SCN1B Convulsions, benign familial infantile, 3 1 SCN2A1 Brugada syndrome 7 1 SCN3B Myotonia congenita, atypical, acetazolamide-responsive 1 SCN4A Paramyotonia congenita 1 SCN4A Long QT syndrome-10 1 SCN4B Brugada syndrome 1 1 SCN5A Cardiomyopathy, dilated, I E 1 SCN5A Heart block, nonprogressive 1 SCN5A Heart block, progressive, type IA 1 SCN5A Long QT syndrome-3 1 SCN5A Ventricular fibrillation, familial, 1 1 SCN5A

4 2

Table IX-21781338.1 Erythermalgia, primary 1 SCN9A Febrile convulsions, familial, 3 B 1 SCN9A Insensitivity to pain, channelopathy-associated 1 SCN9A Bronchiectasis with or without elevated sweat chloride 2 1 SCN N1A Bronchiectasis with o r without elevated sweat chloride 1 1 SCN N1B Bronchiectasis with o r without elevated sweat chloride 3 1 SCNN1G Cardioencephalomyopathy, fatal infantile, due t o cytochrome c oxidase deficiency 1 SC02 Geroderma osteodysplasticum 1 SCYL1BP1 Cardiomyopathy, dilated, 1GG 1 SDHA Mitochondrial respiratory chain complex II deficiency 1 SDHA Mitochondrial complex II deficiency 1 SDHAF1 Paragangliomas 2 1 SDHAF2 Cowden-like syndrome 2 SDHB Paraganglioma and gastric stromal sarcoma 3 SDHB Paraganglioma, familial chromaffin, 4 1 SDHB Pheochromocytoma 5 SDHB Paraganglioma and gastric stromal sarcoma 3 SDHC Paragangliomas, familial nonchromaffin, 3 1 SDHC Cowden-like syndrome 2 SDHD Paraganglioma and gastric stromal sarcoma 3 SDHD Paragangliomas, familial nonchromaffin, 1, with or without deafness 1 SDHD Pheochromocytoma 5 SDHD Anemia, dyserythropoietic congenital, type II 1 SEC23B Polycystic liver disease 2 SEC63 Thyroid hormone meta bolism, abnormal 1 SECISBP2 Muscular dystrophy, rigid spine, 1 1 SEPN1 Emphysema due t o AAT deficiency 1 SERPINA1 Emphysema-cirrhosis, due t o AAT deficiency 1 SERPINA1 Deafness, autosomal recessive 9 1 1 SERPI NB6 Encephalopathy, familial, with neuroserpin inclusion bodies 1 SERPI NI1 Ataxia-ocular apraxia-2 1 SETX Pulmonary fibrosis, idiopathic 1 SFTPA2 Surfactant metabolism dysfunction, pulmonary, 1 1 SFTPB Surfactant metabolism dysfunction, pulmonary, 2 1 SFTPC Muscular dystrophy, limb-girdle, type 2D 1 SGCA Muscular dystrophy, limb-girdle, type 2E 1 SGCB Cardiomyopathy, dilated, 1L 1 SGCD Muscular dystrophy, limb-girdle, type 2F 1 SGCD Dystonia-11, myoclonic 1 SGCE Muscular dystrophy, limb-girdle, type 2C 1 SGCG

Table IX-21781338.1 Lymphoproliferative syndrome, X-linked 1 SH2D1A Leukemia, acute myeloid 14 SH3GL1 Mononeuropathy of the median nerve, mild 1 SH3TC2 Chromosome 22ql3.3 deletion syndrome 1 SHANK3 Coloboma, ocular 2 SHH Holoprosencephaly-3 1 SHH Microphthalmia, isolated, with coloboma 5 1 SHH Leri-Weill dyschondrosteosis 2 SHOX Short stature, idiopathic familial 2 SHOX Langer mesomelic dysplasia 2 SHOXY Leri-Weill dyschondrosteosis 2 SHOXY Short stature, idiopathic familial 2 SHOXY Stocco dos Santos X-linked mental retardation syndrome 1 SHROOM4 Amish infantile epilepsy syndrome 1 SIAT9 Obesity, severe 2 SIM 1 Brachiootic syndrome 3 1 SIX1 Deafness, autosomal dominant 23 1 SIX1 Holoprosencephaly-2 1 SIX3 Branchiootorenal syndrome 2 1 SIX5 Microphthalmia, isolated, with cataract 2 1 SIX6 Bile acid malabsorption, primary 1 SLC10A2 Bartter syndrome, type 1 1 SLC12A1 Agenesis of the corpus callosum with peripheral neuropathy 1 SLC12A6 Erythrocyte lactate transporter defect 1 SLC16A1 Hyperinsulinemic hypoglycemia, familial, 7 1 SLC16A1 Cataract, juvenile, with microcornea and glucosuria 1 SLC16A12 Sialic acid storage disorder, infantile 1 SLC17A5 Deafness, autosomal dominant 25 1 SLC17A8 Basal ganglia disease, biotin-responsive 1 SLC19A3 Dicarboxylicaminoaciduria 1 SLC1A1 Episodic ataxia, type 6 1 SLCIA3 Hypouricemia, renal 1 SLC22A12 Breast cancer 3 SLC22A1L Lung cancer 5 SLC22A1L Rhabdomyosarcoma 1 SLC22A1L Carnitine deficiency, systemic primary 1 SLC22A5 Hypomyelination, global cerebral 1 SLC25A12 Microcephaly, Amish type 1 SLC25A19 Epilepsy, neonatal myoclonic, with suppression-burst pattern 1 SLC25A22 Micochondrial phosphate carrier deficiency 1 SLC25A3 Anemia, sideroblastic, pyridoxine-refractory, autosomal recessive 1 SLC25A38

Table IX-21781338.1 Cardiomyopathy, familial hypertrophic 2 SLC25A4 Progressive external ophthalmoplegia with mitochondrial DNA deletions 3 1 SLC25A4 Achondrogenesis lb 1 SLC26A2 De la Chapelle dysplasia 1 SLC26A2 Diastrophic dysplasia, broad bone-platyspondylic variant 1 SLC26A2 Epiphyseal dysplasia, multiple, 4 1 SLC26A2 Chloride diarrhea, congenital, Finnish type 1 SLC26A3 Enlarged vestibular aqueduct 2 SLC26A4 Hyperpigmentation, cutaneous, with hypertrichosis, hepatosplenomegaly, heart anomalies, hearing loss, and hypogonadism 1 SLC29A3 GLUT1 deficiency syndrome 1 1 SLC2A1 GLUT1 deficiency syndrome 2 1 SLC2A1 Fanconi-Bickel syndrome 1 SLC2A2 Hypouricemia, renal, 2 1 SLC2A9 Spastic paraplegia-42 1 SLC33A1 Fanconi renotubular syndrome 2 1 SLC34A1 Nephrolithiasis/osteoporosis, hypophosphatemia 1 1 SLC34A1 Pulmonary alveolar microlithiasis 1 SLC34A2 Testicular microlithiasis 1 SLC34A2 Hypophosphatemic rickets with hypercalciuria 1 SLC34A3 Congenital disorder of glycosylation, type llf 1 SLC35A1 Congenital disorder of glycosylation type lie 1 SLC35C1 Hyperglycinuria 3 SLC36A2 Spondylocheirodysplasia, Ehlers-Danlos syndrome-like 1 SLC39A13 Folate malabsorption, hereditary 1 SLC46A1 Renal tubular acidosis, distal, AD 1 SLC4A1 Renal tubular acidosis, distal, A R 1 SLC4A1 Spherocytosis, type 4 1 SLC4A1 Corneal dystrophy, Fuchs endothelial, 4 1 SLC4A11 Corneal endothelial dystrophy 2 1 SLC4A1 1 Corneal endothelial dystrophy and perceptive deafness 1 SLC4A1 1 Renal tubular acidosis, proximal, with ocular abnormalities 1 SLC4A4 Thyroid dyshormonogenesis 1 1 SLC5A5 Hyperglycinuria 3 SLC6A19 Orthostatic intolerance 1 SLC6A2 Hyperglycinuria 3 SLC6A20 Parkinsonism-dystonia, infantile 1 SLC6A3 Anxiety-related personality traits 1 SLC6A4 Hyperekplexia 2 SLC6A5 Creatine deficiency syndrome, X-linked 1 SLC6A8

Table IX-21781338.1 Nephrolithiasis/osteoporosis, hypophosphatemia 2 1 SLC9A3R1 Mental retardation, X-linked syndromic, Christianson type 1 SLC9A6 Tourette syndrome 1 SLITRK1 Trichotillomania 1 SLITRK1 Meleda disease 1 SLURP1 Rha bdoid tumor predisposition syndrome 2 1 SMARCA4 Rhabdoid predisposition syndrome 1 1 SMARCB1 Spinal muscular atrophy-1 1 SMN1 Spinal muscular atrophy-2 1 SMN1 Spinal muscular atrophy-3 1 SMN 1 Spinal muscular atrophy-4 1 SMN 1 Mental retardation, X-linked, Snyder-Robinson type 1 SMS Cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma syndrome 1 SNAP29 Dementia, Lewy body 1 SNCA Parkinson disease 4 1 SNCA Dementia, Lewy body 1 SNCB Long QT syndrome 12 1 SNT1 Fibromatosis, gingival 1 SOS1 Van Buchem disease 1 SOST PCWH syndrome 1 SOX10 Hypotrichosis-lymphedema-telangiectasia syndrome 1 SOX18 Microphthalmia, syndromic 3 1 SOX2 Optic nerve hypoplasia and abnormalities of the central nervous system 1 SOX2 Mental retardation, X-linked, with isolated growth hormone deficiency 1 SOX3 Acampomelic campomelic dysplasia 1 SOX9 Campomelic dysplasia with autosomal sex reversal 1 SOX9 Spastic paraplegia-4 1 S AST Globozoospermia 2 SPATA16 Spastic paraplegia-11 1 SPG11 Troyer syndrome 1 SPG20 Spastic paraplegia-3A 1 SPG3A Pancreatitis, hereditary 1 SPINK1 Tropical calcific pancreatitis 1 SPIN K1 Atopy 1 SPIN K5 Diarrhea 3, secretory sodium, congenital, syndromic 1 SPINT2 Dystonia, dopa-responsive, due t o sepiapterin reductase deficiency 1 SPR Legius syndrome 1 SPRED1 Elliptocytosis-2 1 SPTA1 Pyropoikilocytosis 1 SPTA1 Spherocytosis, type 3 1 SPTA1

Table IX-21781338.1 Epileptic encephalopathy, early infantile, 5 1 SPTAN1 Neuropathy, hereditary sensory and autonomic, type 1 1 SPTLC1 Neuropathy, hereditary sensory/autonomic, type IC 1 SPTLC2 Paget disease of bone 3 SQSTM1 Pseudovaginal perineoscrotal hypospadias 1 SRD5A2 Congenital disorder of glycosylation, type Ip 1 SRD5A3 Somatostatin analog, resistance to 1 SSTR5 Ichthyosis with hypotrichosis 1 ST14 Lipoid adrenal hyperplasia 1 STAR Mycobacterial infection, atypical, familial disseminated 2 STAT1 Hyper-lgE recurrent infection syndrome 1 STAT3 Growth hormone insensitivity with immunodeficiency 1 STAT5B Microcephaly, primary autosomal recessive, 7 1 STIL Immune dysfunction, with T-cell inactivation due t o calcium entry defect 2 1 STIM1 · Testicular tumor, sporadic 1 STK11 Male infertility with large-headed, multiflagellar, polyploid spermatozoa 1 STK13 Microphthalmia, syndromic 9 1 STRA6 Polyhydramnios, megalencephaly, and symptomatic epilepsy 1 STRADA Deafness, autosomal recessive 16 1 STRC Ichthyosis, x-linked 1 STS Hemophagocytic lymphohistiocytosis, familial, 4 1 STX11 Epileptic encephalopathy, early infantile, 4 1 STXBP1 Mitochondrial DNA depletion syndrome, encephalomyopathic form, with methylmalonic aciduria 1 SUCLA2 Lactic acidosis, fatal infantile 1 SUCLG1 Azoospermia due to perturbations of meiosis 1 SYCP3 Epilepsy, X-linked, with variable learning disabilities and behavior disorders 1 SYN 1 Emery-Dreifuss muscular dystrophy 4 1 SYNE1 Emery-Dreifuss muscular dystrophy 5 1 SYNE2 Mental retardation, autosomal dominant 5 1 SYNGAP Mental retardation, X-linked, with o r without epilepsy 1 SYP Congenital heart disease, nonsyndromic, 2 1 TAB2 Hypogonadotropic hypogonadism 6 TAC3 Hypogonadotropic hypogonadism 6 TACR3 Corneal dystrophy, gelatinous drop-like 1 TACSTD2 Dystonia-Parkinsonism, X-linked 1 TAF1 Chondrosarcoma, extraskeletal myxoid 3 TAF15 Bare lymphocyte syndrome, type 1 2 TAP1

Bare lymphocyte syndrome, type 1, due to TAP2 deficiency 1 TAP2 Bare lymphocyte syndrome, type 1 2 TAPBP 47

Table IX-21781338.1 Frontotemporal lobar degeneration, TARDBP-related 1 TARDBP Barth syndrome 1 TAZ Cardiomyopathy, dilated, 3A 1 TAZ Hypoparathyroidism-retardation-dysmorphism syndrome 1 TBCE Kenny-Caffey syndrome-1 1 TBCE Spinocerebellar ataxia 17 1 TBP Adrenocorticotropic hormone deficiency 1 TBS19 Conotruncal anomaly face syndrome 1 TBX1 DiGeorge syndrome 1 TBX1 Velocardiofacial syndrome 1 TBX1 Cousin syndrome 1 TBX15 Asthma and nasal polyps 1 TBX21 Cleft palate with ankyloglossia 1 TBX22 Small patella syndrome 1 TBX4 Cardiomyopathy, dilated, I N 1 TCAP Muscular dystrophy, limb-girdle, type 2G 1 TCAP Osteopetrosis, recessive 1 1 TCIRG 1 Treacher Collins mandibulofacial dysostosis 1 TCOF1 Sveinsson choreoretinal atrophy 1 TEAD1 Deafness, autosomal dominant 8/12 1 TECTA Deafness, autosomal recessive 21 1 TECTA Venous malformations, multiple cutaneous and mucosal 1 TEK Aplastic anemia 1 TERC Atransferrinemia · 1 TF Renal cell carcinoma, papillary, 1 2 TFE3 Chondrosarcoma, extraskeletal myxoid 3 TFG Thyroid dyshormonogenesis 3 1 TG Arrhythmogenic right ventricular dysplasia 1 1 TGFB3 Corneal dystrophy, Avellino type 1 TGFBI Corneal dystrophy, epithelial basement membrane 1 TGFBI Corneal dystrophy, Groenouw type 1 1 TGFBI Corneal dystrophy, lattice type 1 1 TGFBI Corneal dystrophy, lattice type IMA 1 TGFBI Corneal dystrophy, Reis-Bucklers type 1 TGFBI Corneal dystrophy, Thiel-Behnke type 1 TGFBI Esophageal cancer, somatic 1 TGFBR2 Holoprosencephaly-4 1 TGI F lchthyosiform erythroderma, congenital 1 TGM1 Ichthyosis, lamellar, autosomal recessive 1 TGM1 Peeling skin syndrome, acral type 1 TGM6 Segawa syndrome, recessive 1 TH

Table IX-21781338.1 Dystonia 6, torsion 1 THAP1 Thrombocythemia, essential 3 THPO Thyroid hormone resistance 1 TH B Thyroid hormone resistance, autosomal recessive . 1 TH RB Thyroid hormone resistance, selective pituitary 1 THRB Jensen syndrome 1 TIMM8A evesz syndrome 1 TINF2 Hypercholanemia, familial 3 TJ P2 Mental retardation, X-linked 58 1 TM4SF2 Deafness, autosomal dominant 36 1 TMC1 Deafness, autosomal recessive 7 1 TMC1 Optic atrophy-7 1 TMEM126A Congenital disorder of glycosylation, type Im 1 TMEM15 Arrhythmogenic right ventricular dysplasia, familial, 5 1 TMEM43 COACH syndrome 3 TMEM67 Encephalocardiomyopathy, neonatal, mitochondrial, due to ATP synthase deficiency 1 TM EM70 Deafness, autosomal recessive 6 1 TM IE Deafness, autosomal recessive 10, congenital 1 TMPRSS3 Deafness, autosomal recessive 8, childhood onset 1 TMPRSS3 Iron-refractory iron deficiency anemia 1 TMPRSS6 Osteolysis, familial expansile 1 TNFRSF11A Osteopetrosis, autosomal recessive 7 1 TNFRSFllA Paget disease of bone 3 TNFRSF11A Paget disease, juvenile 1 TNFRSF11B Immunodeficiency, common variable, 2 1 TN FRSF13B Immunoglobulin A deficiency 2 1 TN FRSF13B Immunodeficiency, common variable, 4 1 TN FRSF13C Periodic fever, familial 1 TNFRSF1A Immunodeficiency with hyper-lgM, type 3 1 TNFRSF5 Autoimmune lymphoproliferative syndrome, type IA 1 TNFRSF6 Osteopetrosis, autosomal recessive 2 1 TNFSF11 Immunodeficiency, X-linked, with hyper-lgM 1 TNFSF5 Cardiomyopathy, dilated, 1Z 1 TNNC1 Cardiomyopathy, familial hypertrophic, 13 1 TNNC1 Arthrogryposis multiplex congenita, distal type 2B 1 TNNI2 Cardiomyopathy, dilated, IFF 1 TNNI3 Cardiomyopathy, dilated, 2A 1 TNNI3 Cardiomyopathy, familial restrictive 1 TNNI3 Nemaline Myopathy, Amish Type 1 TNNT1 Cardiomyopathy, dilated, I D 1 TNNT2

Table IX-21781338.1 Cardiomyopathy, familial hypertrophic, 2 1 TNNT2 Cardiomyopathy, familial restrictive, 3 1 TNNT2 Arthyrgryposis, distal, type 2B 1 T NT3 Adrenal cortical carcinoma 1 TP53 Breast cancer 3 TP53 Choroid plexus papilloma 1 TP53 Colorectal cancer 6 TP53 Hepatocellular carcinoma 2 TP53 Osteosarcoma 3 TP53 Pancreatic cancer 2 TP53 ADULT syndrome 1 TP63 Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3 1 TP63 Hay-Wells syndrome 1 TP63 Orofacial cleft 8 1 TP63 Cardiomyopathy, dilated, 1Y 1 TPM1 Cardiomyopathy, familial hypertrophic, 3 1 TPM1 Arthrogryposis multiplex congenita, distal type 1 1 TPM2 Arthrogryposis, distal, type 2B 2 TPM2 Nemaline myopathy 1 TPM2 Nemaline myopathy 1, autosomal dominant 1 TPM3 6-mercaptopurine sensitivity 1 T T Thyroid dyshormonogenesis 2A 1 TPO Ceroid-lipofuscinosis, neuronal 2, classic late infantile 1 TPP1 Deafness, autosomal recessive 79 1 TP N Mental retardation, autosomal recessive 13 1 TRAPPC9 Trehalase deficiency 1 TREH Nasu-Hakola disease 2 TREM2 Vasculopathy, retinal, with cerebral leukodystrophy 1 TREX1 Thyroid carcinoma, papillary 6 TRIM24 Muscular dystrophy, limb-girdle, type 2H 1 TRIM32 Thyroid carcinoma, papillary 6 TRIM33 Deafness, autosomal recessive 28 1 TRIOBP Liver failure, acute infantile 1 TRMU Glomerulosclerosis, focal segmental, 2 1 TRPC6 Night blindness, congenital stationary, type IC 1 TRPM1 Progressive familial heart block, type IB 1 TRPM4 Hypomagnesemia with secondary hypocalcemia 1 TRPM6 Hereditary motor and sensory neuropathy, type lie 1 TRPV4 Focal cortical dysplasia, Taylor balloon cell type 1 TSC1 Combined oxidative phosphorylation deficiency 3 1 TSFM Hypothryoidism, congenital, nongoitrous 4 1 TSH B

Table IX-21781338.1 Hyperthyroidism, familial gestational 1 TSHR Hyperthyroidism, nonautoimmune 1 TSHR Hypothyroidism, congenital, nongoitrous 1 TSHR Exudative vitreoretinopathy 5 1 TSPAN12 Muscular dystrophy, limb-girdle, type 1A . 1 TTID Myopathy, spheroid body 1 TTID Myotilinopathy 1 TTID Cardiomyopathy, dilated, 1G 1 TTN Muscular dystrophy, limb-girdle, type 2J 1 TTN Myopathy, early-onset, with fatal cardiomyopathy 1 TTN Myopathy, proximal, with early respiratory muscle involvement 1 TTN Amyloidosis, hereditary, transthyretin-related 1 TTR Carpal tunnel syndrome, familial 1 TTR Lissencephaly 3 1 TUBA1A Macrothrombocytopenia, autosomal dominant, TUBBl-related 1 TUBB1 Fibrosis of extraocular muscles, congenital, 3A 1 TUBB3 Combined oxidative phosphorylation deficiency 4 1 TUFM Mental retardation, autosomal recessive 7 1 TUSC3 Craniosynostosis, type 1 1 TWIST1 Saethre-Chotzen syndrome 2 TWIST1 Saethre-Chotzen syndrome with eyelid anomalies 1 TWISTl Ciliary dyskinesia, primary, 6 1 TXN DC3 Tyrosine kinase 2 deficiency 1 TYK2 Albinism, oculocutaneous, type IA 1 TYR Albinism, oculocutaneous, type IB 1 TYR Nasu-Hakola disease 2 TYROBP Albinism, brown 1 TYRP1 Albinism, rufous 1 TYRP1 Cleft palate, isolated 1 UBB Spinal muscular atrophy, X-linked 2, infantile 1 UBE1 Corneal dystrophy, crystalline, of Schnyder 1 UBIAD1 Hyperbilirubinemia, familial transcient neonatal 1 UGT1A1 Glomerulocystic kidney disease with hyperuricemia and isosthenuria 1 UMOD Hyperuricemic nephropathy, familial juvenile 1 1 UMOD Medullary cystic kidney disease 2 1 UMOD Hemophagocytic lymphohistiocytosis, familial, 3 1 UNC13D Immunodeficiency with hyper IgM, type 4 1 UNG Mental retardation, X-linked, syndromic 14 1 UPF3B Renal adysplasia 1 UPK3A Mitochondrial complex III deficiency 1 UQCRB Mitochondrial complex III deficiency 1 UQCRQ

5 1

Table IX-21781338.1 Porphyria, hepatoerythropoietic 1 UROD Porphyria, congenital erythropoietic 1 UROS Deafness, autosomal recessive 18 1 USH1C Azoospermia 1 USP9Y Caudal regression syndrome 1 VANGL1 Neural tube defects 1 VANGL1 Spinal muscular atrophy, late-onset, Finkel type 1 VAPB Cardiomyopathy, dilated, 1W 1 VCL Cardiomyopathy, familial hypertrophic, 15 1 VCL Osteoporosis, involutional 1 VDR Rickets, vitamin D-resistant, type HA 1 VDR Pheochromocytoma 5 VHL Polycythemia, benign familial 1 VHL Renal cell carcinoma, somatic 1 VHL Arthrogryposis, renal dysfunction, and cholestasis 2 1 VIPAR Vitamin K-dependent clotting factors, combined deficiency of, 2 1 VKORC1 Warfarin resistance 1 VKORC1 Cerebellar hypoplasia and mental retardation with or without quadrupedal locomotion 1 1 VLDLR Arthrogryposis, renal dysfunction, and cholestasis 1 1 VPS33B Corneal dystrophy, hereditary polymorphous posterior 1 VSX1 Keratoconus 1 VSX1 Neutropenia, severe congenital, X-linked 1 WAS Thrombocytopenia, X-linked 1 WAS Thrombocytopenia, X-linked, intermittent 1 WAS Glaucoma 1, open angle, G 1 WDR36 Amelogenesis imperfecta, hypomaturation type, IIA3 1 WDR72 Hearing loss, low-frequency sensorineural 1 WFS1 Wolfram syndrome 1 WFS1 Wolfram-like syndrome, autosomal dominant 1 WFS1 Deafness, autosomal recessive 31 1 WHRN Leukemia, acute myeloid 14 WHSC1L1 Arthropathy, progressive pseudorheumatoid, of childhood 1 WISP3 Spondyloepiphyseal dysplasia tarda with progressive arthropathy 1 WISP3 Neuropathy, hereditary sensory and autonomic, type II 1 WNK1 Mullerian aplasia and hyperandrogenism 1 WNT4 Ulna and fibula, absence of, with sever limb deficiency 1 WNT7A Nephrotic syndrome, type 4 1 WT1 Wilms tumor, type 1 1 WT1 Esophageal squamous cell carcinoma 2 WWOX Xanthinuria, type 1 1 XDH

Table IX-21781338.1 X-inactivation, familial skewed 1 IC Charcot-Marie-Tooth disease, dominant intermediate C 1 YARS Skeletal defects, genital hypoplasia, and mental retardation 1 ZBTB16 Mental retardation, X-linked-91 1 ZDHHC15 Mental retardation, X-linked, ZDHHC9-related 1 ZDHHC9 Corneal dystrophy, Fuchs endothelial, 6 1 ZEB1 Corneal dystrophy, posterior polymorphous, 3 1 ZEB1 Mowat-Wilson syndrome 1 ZEB2 Ptosis, congenital 1 ZFHX4 Diabetes mellitus, transient neonatal, 1 1 ZFP57 Diaphragmatic hernia 3 1 ZFPM2 Spastic paraplegia 15 1 ZFYVE26 Spastic paraplegia 33 1 ZFYVE27 Dandy-Walker malformation 1 ZIC1 Holoprosencephaly-5 1 ZIC2 Congenital heart defects, nonsyndromic, 1, X-linked 1 ZIC3 Heterotaxy, visceral, 1, S-linke 1 ZIC3 Dandy-Walker malformation 1 ZIC4 Restrictive dermopathy, lethal 1 ZMPSTE24 Sertoli-cell-only syndrome 1 ZN F148 Mental retardation, X-linked 45 1 ZNF81 Myotonic dystrophy, type 2 1 ZNF9 Triple A syndrome 1 AAAS Punctate palmoplanar keratoderma type 1 2 AAGAB Autosomal dominant Charcot-Marie-Tooth disease type 2N 1 AARS Combined oxidative phosphorylation defect type 8 1 AARS2 Hyperlysinemia 1 AASS Saccharopinuria 1 AASS Gamma aminobutyric acid transaminase deficiency 1 ABAT Apolipoprotein A-l deficiency 2 ABCA1 Tangier disease 1 ABCA1 Congenital nonbullous ichthyosiform erythroderma 6 ABCA12 Harlequin ichthyosis 1 ABCA12 Lamellar ichthyosis 6 ABCA12 Congenital pulmonary alveolar proteinosis 5 ABCA3 Neonatal acute respiratory distress with surfactant metabolism deficiency 2 ABCA3 Retinitis pigmentosa 61 ABCA4 Stargardt disease 3 ABCA4 Benign recurrent intrahepatic cholestasis type 2 1 ABCB11 Intrahepatic cholestasis of pregnancy 4 ABCB11 Progressive familial intrahepatic cholestasis type 2 1 ABCB11

Table IX-21781338.1 Intrahepatic cholestasis of pregnancy 4 ABCB4 Low phospholipid associated cholelithiasis 1 ABCB4 Progressive familial intrahepatic cholestasis type 3 1 ABCB4 Colobomatous microphthalmia 8 ABCB6 X-linked sideroblastic anemia -' ataxia 1 ABCB7 Dubin-Johnson syndrome 1 ABCC2 Generalized arterial calcification of infancy 2 ABCC6 Pseudoxanthoma elasticum 1 ABCC6 Autosomal dominant hyperinsulinism due to SURl deficiency 1 ABCC8 Autosomal recessive hyperinsulinism due to SURl deficiency 1 ABCC8 MODY syndrome 12 ABCC8 Permanent neonatal diabetes mellitus 5 ABCC8 Transient neonatal diabetes mellitus . 5 ABCC8 Acromegaloid facial appearance syndrome 1 ABCC9 Cantu syndrome 1 ABCC9 Familial atrial fibrillation 14 ABCC9 Familial isolated dilated cardiomyopathy 38 ABCC9 Hypertrichosis with acromegaloid facial appearence 1 ABCC9 Adrenomyeloneuropathy 1 ABCD1 X-linked cerebral adrenoleukodystrophy 1 ABCD1 Methylmalonic acidemia with homocystinuria, type cbl F 2 ABCD4 Sitosterolemia 2 ABCG5 Sitosterolemia 2 ABCG8 Polyneuropathy - hearing loss - ataxia - retinitis pigmentosa - cataract 1 ABHD12 Dorfman-Chanarin disease 1 ABHD5 Chronic myeloid leukemia 3 ABL1 Precursor B-cell acute lymphoblastic leukemia 13 ABL1 Precursor T-cell acute lymphoblastic leukemia 19 ABL1 Isobutyryl-CoA dehydrogenase deficiency 1 ACAD8 Acyl-CoA dehydrogenase 9 deficiency 1 ACAD9 Isolated NADH-CoQ reductase deficiency 25 ACAD9 Long chain acyl-CoA dehydrogenase deficiency 1 ACADL Medium chain acyl-CoA dehydrogenase deficiency 1 ACADM Short chain acyl-CoA dehydrogenase deficiency 1 ACADS 2-methylbutyryl-CoA dehydrogenase deficiency 1 ACADSB Very long chain acyl-CoA dehydrogenase deficiency 1 ACADVL Familial osteochondritis dissecans 1 ACAN Spondyloepimetaphyseal dysplasia, aggrecan type 1 ACAN Spondyloepiphyseal dysplasia, Kim berley type 1 ACAN Ketoacidosis due t o betaketothiolase deficiency 1 ACAT1 Renal tubular dysgenesis of genetic origin 4 ACE

Table IX-21781338.1 Infantile cerebellar-retinal degeneration 1 AC02 Peroxisomal acyl-CoA oxidase deficiency 1 ACOX1 Acid phosphatase deficiency 1 ACP2 Spondyloenchondrodysplasia 1 ACP5 Combined malonic and methylmalonic acidemia 1 ACSF3 X-linked nonsyndromic intellectual deficit 24 ACSL4 Childhood-onset nemaline myopathy 5 ACTA1 Congenital myopathy with excess of thin filaments 1 ACTA1 Intermediate nemaline myopathy 3 ACTA1 Severe congenital nemaline myopathy 3 ACTA1 Typical nemaline myopathy 4 ACTA1 Familial thoracic aortic aneurysm and aortic dissection 8 ACTA2 Moyamoya disease 2 ACTA2 Baraitser-Winter syndrome 2 ACTB Developmental malformations - deafness - dystonia 1 ACTB Atrial septal defect, ostium secundum type 8 ACTC1 Familial isolated dilated cardiomyopathy 38 ACTC1 Left ventricular noncompaction 11 ACTC1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 ACTG1 Baraitser-Winter syndrome 2 ACTG1 Familial visceral myopathy 1 ACTG2 Autosomal dominant macrothrombocytopenia 3 ACTN1 Familial isolated dilated cardiomyopathy 38 ACT 2 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 ACTN4 Fibrodysplasia ossificans progressiva 1 ACVR1 Situs ambiguus 6 ACVR2B Heritable pulmonary arterial hypertension 5 ACVRL1 Rendu-Osler-Weber disease 3 ACVRL1 Neurological conditions associated with aminoacylase 1 deficiency 1 ACY1 Omenn syndrome 9 ADA Severe combined immunodeficiency due to adenosine deaminase deficiency 1 ADA Reticulate acropigmentation of Kitamura 1 ADAM10 Neonatal inflammatory skin and bowel disease 1 ADAM17 Cone rod dystrophy 22 ADAM9 Weill-Marchesani syndrome 4 ADAMTS10 Congenital thrombotic thrombocytopenic purpura due to ADAMTS-13 deficiency 1 ADAMTS13 Weill-Marchesani syndrome 4 ADAMTS17 Ehlers-Danlos syndrome, dermatosparaxis type 1 ADAMTS2 Geleophysic dysplasia 2 ADAMTSL2 55

Table IX-21781338.1 Ectopia lentis syndrome 2 ADAMTSL4 Aicardi-Goutieres syndrome 6 ADAR Dyschromatosis symmetrica hereditaria 1 ADAR Autosomal recessive ataxia due to ubiquinone deficiency 1 ADCK3 Idiopathic hypercalciuria 1 ADCY10 Familial dyskinesia and facial myokymia 1 ADCY5 Hypermethioninemia encephalopathy due to adenosine kinase deficiency 1 ADK Adenylosuccinate lyase deficiency 1 ADSL Precursor B-cell acute lymphoblastic leukemia 13 AFF1 F A XE intellectual deficit 2 AFF2 Early-onset spastic ataxia-neuropathy syndrome 1 AFG3L2 Spinocerebellar ataxia type 28 1 AFG3L2 Congenital deficiency in alpha-fetoprotein 1 AFP Hereditary persistence of alpha-fetoprotein 1 AFP Aspartylglucosaminuria 1 AGA Klippel-Trenaunay syndrome 1 AGGF1 Congenital cataract - hypertrophic cardiomyopathy - mitochondrial myopathy 2 AGK Berardinelli-Seip congenital lipodystrophy 3 AG AT2 Rhizomelic chondrodysplasia punctata type 3 1 AGPS Postsynaptic congenital myasthenic syndromes 9 AGRN Renal tubular dysgenesis of genetic origin 4 AGT Renal tubular dysgenesis of genetic origin 4 AGTR1 X-linked nonsyndromic intellectual deficit 24 AGTR2 Primary hyperoxaluria type 1 1 AGXT Psychomotor retardation due t o S-adenosylhomocysteine hydrolase deficiency 1 AHCY Joubert syndrome with ocular defect 5 AHI1 Hyper-lgM syndrome type 2 1 AICDA Severe X-linked mitochondrial encephalomyopathy 1 A IFM1 X-linked Charcot-Marie-Tooth disease type 4 1 AIFM1 Pelizaeus-Merzbacher-like due to A IMP1 mutation 1 AIMP1 Acromegaly 2 AIP Familial isolated pituitary adenoma 1 A IP Cone rod dystrophy 22 AIPL1 Leber congenital amaurosis 18 AIPL1 Retinitis pigmentosa 6 1 AIPL1 Autoimmune polyendocrinopathy type 1 1 A IRE Hemolytic anemia due t o adenylate kinase deficiency 1 AK1 Reticular dysgenesis 1 AK2 Romano-Ward syndrome 13 AKAP9

Table IX-21781338.1 46,XY disorder of sex development due to isolated 17, 20 lyase deficiency 4 AKR1C4 Congenital bile acid synthesis defect type 2 1 AKR1D1 Cowden syndrome 7 AKT1 Proteus syndrome 2 AKT1 Familial partial lipodystrophy due to AKT2 mutations 1 AKT2 Hypoinsulinemic hypoglycemia and body hemihypertrophy 1 AKT2 Hemimegalencephaly 2 AKT3 Megalencephaly - polymicrogyria - post-axial Polydactyly - hydrocephalus 2 AKT3 Porphyria due t o ALA dehydratase deficiency 1 ALAD Erythropoietic protoporphyria 2 ALAS2 X-linked sideroblastic anemia 1 ALAS2 Congenital analbuminemia 1 ALB ALDH18Al-related DeBarsy syndrome 1 ALDH18A1 Isolated anophthalmia - microphthalmia 5 ALDH1A3 Sjogren-Larsson syndrome 1 ALDH3A2 Hyperprolinemia type II 1 ALDH4A1 Developmental delay due to methylmalonate semialdehyde dehydrogenase deficiency 1 ALDH6A1 Pyridoxine-dependent epilepsy 1 ALDH7A1 Glycogen storage disease due to aldolase A deficiency 1 ALDOA Hereditary fructose intolerance 1 ALDOB ALG1-CDG syndrome 1 ALG1 Romano-Ward syndrome 13 ALG10 ALG 11-CDG syndrome 1 ALG 11 ALG 12-CD.G syndrome 1 ALG 12 ALG 13-CDG syndrome 1 ALG 13 Congenital myasthenic syndromes with glycosylation defect 4 ALG 14 ALG2-CDG syndrome 1 ALG2 Congenital myasthenic syndromes with glycosylation defect 4 ALG2 ALG3-CDG syndrome 1 ALG3 ALG6-CDG syndrome 1 ALG6 ALG8-CDG syndrome 1 ALG8 ALG9-CDG syndrome 1 ALG9 ALK-positive anaplastic large cell lymphoma 1 ALK Inflammatory myofibroblastic tumor 6 ALK Neuroblastoma 7 ALK Alstrom syndrome 1 ALMS1 Congenital nonbullous ichthyosiform erythroderma 6 ALOX12B Lamellar ichthyosis 6 ALOX12B Self-healing collodion baby 3 ALOX12B Congenital nonbullous ichthyosiform erythroderma 6 ALOXE3

Table IX-21781338.1 Self-healing collodion baby 3 ALOXE3 Adult hypophosphatasia 1 ALPL Childhood-onset hypophosphatasia 1 ALPL Infantile hypophosphatasia 1 ALPL Odontohypophosphatasia 1 ALPL Perinatal lethal hypophosphatasia 1 ALPL Infantile-onset ascending hereditary spastic paralysis 1 ALS2 Juvenile amyotrophic lateral sclerosis 3 ALS2 Juvenile primary lateral sclerosis 2 ALS2 Frontonasal dysplasia-severe microphthalmia-severe facial defting syndrome 1 ALX1 Frontonasal dysplasia 2 ALX4 Frontonasal dysplasia with alopecia and genital anomaly 1 ALX4 Isolated scaphocephaly 3 ALX4 Parietal foramina 2 ALX4 Potocki-Shaffer syndrome 3 ALX4 Congenital bile acid synthesis defect type 4 1 AMACR Hypomaturation amelogenesis imperfecta 5 AMELX Osteopathia striata - cranial sclerosis 1 AMER1 Persistent Mullerian duct syndrome 2 AMH Persistent Mullerian duct syndrome 2 AMHR2 Alport syndrome - intellectual deficit - midface hypoplasia - elliptocytosis 3 AMMECR1 Grasbeck-lmerslund disease 2 AMN Adenosine monophosphate deaminase deficiency 2 AM PD1 Adenosine monophosphate deaminase deficiency 2 AMPD3 Atypical glycine encephalopathy 3 AMT Infantile glycine encephalopathy 3 AMT Neonatal glycine encephalopathy 3 AMT Amyotrophic lateral sclerosis 23 ANG 8pll. 2 deletion syndrome 1 ANK1 Hereditary spherocytosis 5 ANK1 Romano-Ward syndrome 13 ANK2 Intellectual deficiency - hypotonia - spasticity - sleep disorder 1 ANK3 Craniometaphyseal dysplasia . 1 ANKH Familial articular chondrocalcinosis 1 ANKH 16q24.3 microdeletion syndrome 1 ANKRD11 KBG syndrome 1 ANKRD11 Autosomal thrombocytopenia with normal platelets 3 ANKRD26 Juvenile rheumatoid factor-negative polyarthritis 9 ANKRD55 Oligoarticular juvenile arthritis 9 ANKRD55 Adult-onset autosomal recessive cerebellar ataxia 1 ANO10

Table IX-21781338.1 Cervical dystonia 2 AN03 Autosomal recessive limb-girdle muscular dystrophy type 2L 1 AN05 Miyoshi myopathy 2 AN05 Scott syndrome 1 AN06 Familial capillary hemangioma 2 ANTX 1 GAPO syndrome 1 ANTXR1 Infantile systemic hyalinosis 1 ANTXR2 Juvenile hyaline fibromatosis 1 ANTXR2 M EDNIK syndrome 1 AP1S1 Fried syndrome 1 AP1S2 Familial hypocalciuric hypercalcemia type 3 1 AP2S1 Hermansky-Pudlak syndrome with neutropenia 1 AP3B1 Severe intellectual deficit and progressive spastic paraplegia 4 AP4B1 Severe intellectual deficit and progressive spastic paraplegia 4 AP4E1 Severe intellectual deficit and progressive spastic paraplegia 4 AP4M1 Severe intellectual deficit and progressive spastic paraplegia 4 AP4S1 Autosomal recessive spastic paraplegia type 48 1 AP5Z1 APC-related attenuated familial adenomatous polyposis 1 APC Desmoid disease 2 APC Familial adenomatous polyposis due t o 5q22.2 microdeletion 1 APC Gardner syndrome 1 APC Turcot syndrome with polyposis 1 APC Hypotrichosis simplex 6 APCDD1 Apolipoprotein A-l deficiency 2 APOA1 Familial renal amyloidosis due to Apolipoprotein Al variant 1 APOA1 Primary systemic amyloidosis 1 APOA1 Familial renal amyloidosis due to Apolipoprotein All variant 1 APOA2 Hyperlipoproteinemia type 4 2 APOA5 Hyperlipoproteinemia type 5 4 APOA5 Abetalipoproteinemia 2 APOB Familial apolipoprotein C-ll deficiency 1 APOC2 Cholesterol-ester transfer protein deficiency 2 APOC3 Hyperlipidemia type 3 1 APOE Lipoprotein glomerulopathy 1 APOE Sea-blue histiocytosis 1 APOE Sporadic idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 2 APOL1 Early-onset autosomal dominant Alzheimer disease 4 APP Hereditary cerebral hemorrhage with amyloidosis, Arctic type 1 APP Hereditary cerebral hemorrhage with amyloidosis, Dutch type 1 APP Hereditary cerebral hemorrhage with amyloidosis, Flemish type 1 APP

Table IX-21781338.1 Hereditary cerebral hemorrhage with amyloidosis, Iowa type 1 APP Hereditary cerebral hemorrhage with amyloidosis, Italian type 1 APP Hereditary cerebral hemorrhage with amyloidosis, Piedmont type 1 APP Adenine phosphoribosyltransferase deficiency 1 APRT Ataxia - oculomotor apraxia type 1 1 APTX Nephrogenic diabetes insipidus 2 AQP2 Complete androgen insensitivity syndrome 1 A R Familial hypospadias 2 A R Kennedy disease 1 A R Partial androgen insensitivity syndrome 1 A R Periventricular nodular heterotopia 2 ARFGEF2 Argininemia 1 ARG1 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 ARHGAP24 Adams-Oliver syndrome 4 ARHGAP31 Familial idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial sclerosis 4 ARHGDIA Autosomal dominant slowed nerve conduction velocity 1 ARHGEF10 X-linked nonsyndromic intellectual deficit 24 ARHGEF6 Hyperekplexia - epilepsy 1 ARHGEF9 Coff in-Siris syndrome 4 ARI D1A 6q25 microdeletion syndrome 1 ARID1B Coffin-Siris syndrome 4 ARID1B B-cell chronic lymphocytic leukemia 7 ARL11 Jou bert syndrome 9 ARL13B Bardet-Biedl syndrome 17 ARL6 Retinitis pigmentosa 6 1 ARL6 Metachromatic leukodystrophy, adult form 2 ARSA Metachromatic leukodystrophy, juvenile form 2 AR5A Metachromatic leukodystrophy, late infantile form 2 ARSA Mucopolysaccharidosis type 6, rapidly progressing 1 ARSB Brachytelephalangic chondrodysplasia punctata 1 ARSE 22qll.2 deletion syndrome 7 ARVCF Early infantile epileptic encephalopathy 9 ARX Micrencephaly - corpus callosum agenesis - abnormal genitalia 1 ARX Partington syndrome 1 ARX Spasticity - intellectual deficit - X-linked epilepsy 1 ARX West syndrome 4 ARX X-linked lissencephaly with abnormal genitalia 1 ARX X-linked nonsyndromic intellectual deficit 24 ARX Farber lipogranulomatosis 1 ASAH1

Table IX-21781338.1 Hereditary myoclonus - progressive distal muscular atrophy 1 ASAH1 Haddad syndrome 3 ASCL1 Ondine syndrome . 5 ASCL1 Argininosuccinic aciduria 1 ASL Mild Canavan disease 1 ASPA Severe Canavan disease 1 ASPA Autosomal recessive primary microcephaly 11 ASPM Alveolar soft-part sarcoma 2 ASPSCR1 Translocation renal cell carcinoma 7 ASPSCR1 Acute neonatal citrullinemia type 1 1 ASS1 Adult-onset citrullinemia type 1 1 ASS1 Bohring-Opitz syndrome 1 ASXL1 Severe feeding difficulties - failure to thrive - microcephaly due to ASXL3 deficiency 1 ASXL3 Cerebellar ataxia, Cayman type 1 ATCAY Melanoma of soft part 3 ATF1 AICA-ribosiduria 1 ATIC Autosomal dominant spastic paraplegia type 3 1 ATL1 Hereditary sensory and autonomic neuropathy type 1 4 ATL1 Ataxia-telangiectasia 1 ATM B-cell chronic lymphocytic leukemia 7 ATM Mantle cell lymphoma 3 ATM Dentatorubral-pallidoluysian atrophy 1 ATN1 . Congenital blindness due to retinal nonattachment 1 ATOH7 Congenital cataract microcornea with corneal opacity 2 ATOH7 Persistent hyperplastic primary vitreous 3 ATOH7 Angelman syndrome 6 ATP10A Idiopathic pulmonary fibrosis 11 ATP11A Kufor-Rakeb syndrome 1 ATP13A2 Parkinsonim due to ATP13A2 deficiency 1 ATP13A2 Alternating hemiplegia of childhood 4 ATP1A2 Familial or sporadic hemiplegic migraine 4 ATP1A2 Alternating hemiplegia of childhood 4 ATPI A3 Rapid-onset dystonia-parkinsonism 1 ATPI A3 Brody myopathy 1 ATP2A1 Acrokeratosis verruciformis of Hopf 1 ATP2A2 Darier disease 1 ATP2A2 X-linked non progressive cerebellar ataxia 1 ATP2B3 Familial benign chronic pemphigus 1 ATP2C1 Isolated ATP synthase deficiency 4 ATP5A1 Isolated ATP synthase deficiency 4 ATP5E

6 1

Table IX-21781338.1 Autosomal recessive cutis laxa type 2, classic type 1 ATP6V0A2 Wrinkly skin syndrome 1 ATP6V0A2 Autosomal recessive distal renal tubular acidosis without deafness 1 ATP6V0A4 Autosomal recessive distal renal tubular acidosis with deafness 1 ATP6V1B1 Menkes disease 1 ATP7A Occipital horn syndrome 1 ATP7A X-linked distal spinal muscular atrophy 1 ATP7A Wilson disease 1 ATP7B Dysequilibrium syndrome 4 ATP8A2 Intrahepatic cholestasis of pregnancy 4 ATP8B1 Progressive familial intrahepatic cholestasis type 1 1 ATP8B1 Isolated ATP synthase deficiency 4 ATPAF2 Familial cutaneous telangiectasia and oropharyngeal predisposition cancer syndrome 1 AT Seckel syndrome 6 ATR Seckel syndrome 6 ATRIP Alpha thalassemia - X-linked intellectual deficit syndrome 1 ATRX Alpha-thalassemia - myelodysplastic syndrome 1 ATRX Carpenter-Waziri syndrome 1 ATRX . Chudley-Lowry-Hoar syndrome 1 ATRX Holmes-Gang syndrome 1 ATRX Juberg-Marsidi syndrome 1 ATRX Smith-Fineman-Myers syndrome 1 ATRX Vasquez-Hurst-Sotos syndrome 1 ATRX Spinocerebellar ataxia type 1 1 ATXN1 Joubert syndrome with renal defect 5 ATXN10 Spinocerebellar ataxia type 10 1 ATX 10 Amyotrophic lateral sclerosis 23 ATXN2 Spinocerebellar ataxia type 2 1 ATXN2 Machado-Joseph disease type 1 1 ATXN3 Machado-Joseph disease type 2 1 ATXN3 Machado-Joseph disease type 3 1 ATXN3 Spinocerebellar ataxia type 7 1 ATXN7 Spinocerebellar ataxia type 8 2 ATXN8 Spinocerebellar ataxia type 8 2 ATXN80S 3-methylglutaconic aciduria type 1 1 AUH X-linked hereditary sensory and autonomic neuropathy with deafness 1 AUNX1 Male infertility associated with large-headed multiflagellar polyploid spermatozoa 1 AURKC Hereditary central diabetes insipidus 1 AVP Nephrogenic diabetes insipidus 2 AVPR2

Table IX-21781338.1 Nephrogenic syndrome of inappropriate antidiuresis 1 AVP 2 Oligodontia 10 AXIN2 Oligodontia - cancer predisposition syndrome 1 AXIN2 Autosomal dominant beta2-microglobulinic amyloidosis 1 B2M Muscle eye brain disease 7 B3GALNT2 Walker-Warburg syndrome 13 B3GALNT2 Ehlers-Danlos syndrome, progeroid type 2 B3GALT6 Spondyloepimetaphyseal dysplasia with joint laxity 1 B3GALT6 Peters-plus syndrome 1 B3GALTL Larsen-like syndrome, B3GAT3 type 1 B3GAT3 Walker-Warburg syndrome 13 B3GNT1 Autosomal recessive spastic paraplegia type 26 1 B4GALNT1 B4GALT1-CDG syndrome 1 B4GALT1 Ehlers-Danlos syndrome, progeroid type 2 B4GALT7 Meckel syndrome 13 B9D1 Meckel syndrome \ 13 B9D2 Familial hypercholanemia 3 BAAT Familial isolated dilated cardiomyopathy 38 BAG3 Muscular dystrophy, Selcen type 1 BAG3 Nestor-Guillermo progeria syndrome 1 BANF1 BAPl-related tumor predisposition syndrome 1 BAP1 Hereditary breast and ovarian cancer syndrome 13 BARD1 Williams syndrome 17 BAZ1B Bardet-Biedl syndrome 17 BBS1 Bardet-Biedl syndrome 17 BBS10 Bardet-Biedl syndrome 17 BBS12 Bardet-Biedl syndrome 17 BBS2 Bardet-Biedl syndrome 17 BBS4 Bardet-Biedl syndrome 17 BBS5 Bardet-Biedl syndrome 17 BBS7 Bardet-Biedl syndrome 17 BBS9 Butyrylcholinesterase deficiency 1 BCHE Classic maple syrup urine disease 4 BCKDHA Intermediate maple syrup urine disease 5 BCKDHA Intermittent maple syrup urine disease 4 BCKDHA Classic maple syrup urine disease 4 BCKDHB Intermediate maple syrup urine disease 5 BCKDHB Intermittent maple syrup urine disease 4 BCKDHB Thiamin-responsive maple syrup urine disease 4 BCKDHB Autism-epilepsy syndrome due t o branched chain ketoacid dehydrogenase kinase deficiency 1 BCKDK

Table IX-21781338.1 Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 BCL11A Follicular lymphoma 4 BCL2 Intravascular large B-cell lymphoma 2 BCL2 Follicular lymphoma 4 BCL6 Intravascular large B-cell lymphoma 2 BCL6 Primary mediastinal large B-cell lymphoma 1 BCL6 Williams syndrome 17 BCL7B Hereditary hypercarotenemia and vitamin A deficiency 1 BCMOl Microphthalmia, Lenz type 1 BCOR Oculofaciocardiodental syndrome 1 BCOR Chronic myeloid leukemia 3 BCR Distal 22qll.2 microdeletion syndrome 3 BCR Precursor B-cell acute lymphoblastic leukemia 13 BCR Precursor T-cell acute lymphoblastic leukemia 19 BCR Bjornstad syndrome 1 BCS1L GRACILE syndrome 1 BCS1L Isolated CoQ-cytochrome C reductase deficiency 6 BCS1L Leigh syndrome with nephrotic syndrome 4 BCS1L Renal tubulopathy - encephalopathy - liver failure 1 BCS1L Ondine syndrome 5 BDNF WAGR syndrome 3 BDNF Spinocerebellar ataxia type 31 1 BEA 1 Adult-onset foveomacular vitelliform dystrophy 2 BEST1 Autosomal dominant vitreoretinochoroidopathy 1 BEST1 Best disease 1 BEST1 MRCS syndrome 1 BEST1 Retinitis pigmentosa 6 1 BEST1 Retinopathy, Burgess-Black type 1 BEST1 Autosomal recessive childhood-onset cortical cataract 1 BFSP1 Partial congenital cataract 1 BFSP2 Tibial aplasia - ectrodactyly 1 BHLHA9 Bilateral multicystic renal dysplasia 1 BICC1 Autosomal recessive centronuclear myopathy 1 BIN 1 MALT lymphoma 4 BIRC3 MODY syndrome 12 BLK Autosomal agammaglobulinemia 7 BLNK Hermansky-Pudlak syndrome type 8 1 BLOC1S3 Hyperbiliverdinemia 1 BLVRA Osteogenesis imperfecta type 3 9 BMP1 46,XX gonadal dysgenesis 4 BMP15 Brachydactyly type A2 3 BMP2

Table IX-Z178 338.1 14q22q23 microdeletion syndrome 2 BMP4 Microphthalmia with brain and digit anomalies 1 BMP4 Diaphanospondylodysostosis 1 BMPER Generalized juvenile polyposis/juvenile polyposis coli 3 BMPR1A Hereditary mixed polyposis syndrome 2 BMPR1A Hereditary nonpolyposis colon cancer 11 BMPR1A Juvenile polyposis of infancy 2 BMPR1A Brachydactyly type A2 3 BMPR1B Brachydactyly type C 2 BMPR1B Heritable pulmonary arterial hypertension 5 BMPR2 Pulmonary venoocclusive disease 1 BMPR2 Circumscribed cutaneous aplasia of the vertex 1 BMS1 Fatal multiple mitochondrial dysfunction syndrome 2 BOLA3 Hemolytic anemia due t o diphosphoglycerate mutase deficiency 1 BPGM Cardiofaciocutaneous syndrome 4 BRAF Hairy cell leukemia 1 BRAF Hashimoto-Pritzker syndrome 1 BRAF LEOPARD syndrome 3 BRAF Noonan syndrome 10 BRAF Pilocytic astrocytoma 2 BRAF Familial pancreatic carcinoma 8 BRCA1 Familial prostate cancer 14 BRCA1 Hereditary breast and ovarian cancer syndrome 13 BRCA1 Hereditary breast cancer 4 BRCA1 Hereditary site-specific ovarian cancer syndrome 2 BRCA1 Primary peritoneal carcinoma 1 BRCA1 Familial pancreatic carcinoma 8 BRCA2 Familial prostate cancer 14 BRCA2 Fanconi anemia 16 BRCA2 Hereditary breast and ovarian cancer syndrome 13 BRCA2 Hereditary breast cancer 4 BRCA2 Hereditary site-specific ovarian cancer syndrome 2 BRCA2 Inherited cancer-predisposing syndrome due t o biallelic BRCA2 mutations 1 BRCA2 Moyamoya disease - short stature - facial dysmorphism - hypergonadotropic hypogonadism 1 BRCC3 Fanconi anemia 16 BRIP1 Hereditary breast and ovarian cancer syndrome 13 BRIP1 Autosomal dominant spastic paraplegia type 17 1 BSCL2 Berardinelli-Seip congenital lipodystrophy 3 BSCL2 Distal hereditary motor neuropathy type 5 3 BSCL2 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 BSND

Table IX-21781338.1 Infantile Bartter syndrome with deafness 3 BSND Biotinidase deficiency 1 BTD Short stature due to isolated growth hormone deficiency with X-linked hypogammaglobulinemia 2 BTK X-linked agammaglobulinemia 1 BTK Sarcoidosis 2 BTNL2 Mosaic variegated aneuploidy syndrome 2 BUB1B Oculocutaneous albinism type 5 1 ClOORFll Autosomal dominant progressive external ophthalmoplegia 5 C10ORF2 Infantile onset spinocerebellar ataxia 1 C10ORF2 Sensory ataxic neuropathy - dysarthria - ophthalmoparesis 2 C10ORF2 Temtamy syndrome 1 C120RF57 Autosomal recessive spastic paraplegia type 55 1 ' C120RF65 Combined oxidative phosphorylation defect type 7 1 C120RF65 Dyskeratosis congenita 10 C160RF57 Poikiloderma with neutropenia 1 C160RF57 Neurodegeneration with brain iron accumulation due to C19orfl2 mutation 1 C190RF12 Immunodeficiency due to an early component of complement deficiency 8 C1QA Immunodeficiency due t o an early component of complement deficiency 8 C1QB Immunodeficiency due t o an early component of complement deficiency 8 C1QC Late-onset retinal degeneration 1 C1QTN F5 Immunodeficiency due to an early component of complement deficiency 8 CIS Immunodeficiency due to an early component of complement deficiency 8 C2 Retinitis pigmentosa 61 C20RF71 Atypical hemolytic uremic syndrome with C3 anomaly 1 C3 Complement component 3 deficiency 1 C3 Immunodeficiency due to an early component of complement deficiency 8 C4A Immunodeficiency due to an early component of complement deficiency 8 C4B Hypomaturation amelogenesis imperfecta 5 C40RF26 Immunodeficiency due to a late component of complements deficiency 7 C5 Joubert syndrome 9 C50RF42 Monomelic amyotrophy 2 C50RF42 Immunodeficiency due to a late component of complements deficiency 7 C6 Immunodeficiency due to a late component of complements deficiency 7 C7 Glutaric acidemia type 3 1 C7ORF10 Immunodeficiency due t o a late component of complements deficiency 7 C8A Immunodeficiency due t o a late component of complements deficiency 7 C8B Immunodeficiency due to a late component of complements deficiency 7 C8G Cone rod dystrophy 22 C80RF37 Retinitis pigmentosa 61 C80RF37

Table IX-21781338.1 Leigh syndrome with leukodystrophy 13 C80RF38 Immunodeficiency due to a late component of complements deficiency 7 C9 Amyotrophic lateral sclerosis 23 C90RF72 Frontotemporal dementia with motor neuron disease 3 C90RF72 Osteopetrosis with renal tubular acidosis 1 CA2 Retinitis pigmentosa ' 6 1 CA4 Dysequilibrium syndrome 4 CA8 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 CABP2 Congenital stationary night blindness 12 CABP4 Alternating hemiplegia of childhood 4 CACNA1A Benign paroxysmal torticollis of infancy 1 CACNA1A Familial or sporadic hemiplegic migraine 4 CACNA1A Familial paroxysmal ataxia 1 CACNA1A Spinocerebellar ataxia type 6 1 CACNA1A Brugada syndrome 12 CACNA1C Timothy syndrome 1 CACNA1C Sinoatrial node dysfunction and deafness 1 CACNA1D Aland Island eye disease 1 CACNA1F Cone rod dystrophy 22 CACNA1F Congenital stationary night blindness 12 CACNA1F Childhood absence epilepsy 6 CACNA1H Hypokalemic periodic paralysis 3 CACNA1S Malignant hyperthermia 2 CACNA1S Thyrotoxic periodic paralysis 3 CACNA1S Familial short QT syndrome 4 CACNA2D1 Early infantile epileptic encephalopathy 9 CACNA2D2 Cone rod dystrophy 22 CACNA2D4 Brugada syndrome 12 CACN B2 Episodic ataxia type 5 1 CACN B4 Juvenile myoclonic epilepsy 7 CACNB4 Autosomal dominant nonsyndromic intellectual deficit 15 CACNG2 Catecholaminergic polymorphic ventricular tachycardia ' 4 CALM1 Familial long QT syndrome 2 CALM1 Familial long QT syndrome 2 CALM2 2p21 microdeletion syndrome 4 CAMKMT Atypical hypotonia - cystinuria syndrome 3 CAMKMT Non progressive cerebellar ataxia with intellectual deficit 1 CAMTA1 Autosomal recessive limb girdle muscular dystrophy type 2A 1 CAPN3 Autosomal dominant neovascular inflammatory vitreoretinopathy 1 CAPN5 Persistent polyclonal B-cell lymphocytosis 1 CARD11 Severe combined immunodeficiency due to CARD11 deficiency 1 CARD11

Table IX-21781338.1 Pityriasis rubra pilaris 1 CARD14 Chronic mucocutaneous candidiasis 6 CARD9 Inflammatory myofibroblastic tumor 6 CARS Autosomal recessive primary microcephaly 11 CASC5 X-linked intellectual deficit, Najm type 1 CASK Autoimmune lymphoproliferative syndrome 6 CASP10 Autoimmune lymphoproliferative syndrome with recurrent infections 1 CASP8 Catecholaminergic polymorphic ventricular tachycardia 4 CASQ2 Autosomal dominant hypocalcemia 1 CASR Bartter syndrome with hypocalcemia 1 CASR Familial hypocalciuric hypercalcemia type 1 1 CASR Familial isolated hypoparathyroidism due t o impaired PTH secretion 2 CASR Neonatal severe primary hyperparathyroidism 1 CASR Acatalasemia 1 CAT CATSPERl-related non syndromic male infertility 1 CATSPER1 Deafness-infertility syndrome 2 CATSPER2 Berardinelli-Seip congenital lipodystrophy ' 3 CAV1 Diffuse cutaneous systemic sclerosis 4 CAV1 Heritable pulmonary arterial hypertension 5 CAV1 Limited cutaneous systemic sclerosis 4 CAV1 Autosomal dominant limb-girdle muscular dystrophy type 1C 1 CAV3 Rippling muscle disease 1 CAV3 Romano-Ward syndrome 13 CAV3 Acute megakaryoblastic leukemia without Down syndrome 2 CBFA2T3 Acute myeloid leukemia with abnormal bone marrow eosinophils inv( 16)(pl3q22) or t(16;16)(pl3;q22) 2 CBFB Noonan syndrome 10 CBL Heritable pulmonary arterial hypertension 5 CBLN2 Classical homocystinuria 1 CBS 46,XY complete gonadal dysgenesis 8 CBX2 Autosomal recessive nonsyndromic intellectual deficit 15 CC2D1A Joubert syndrome with hepatic defect 3 CC2D2A Joubert syndrome with oculorenal defect 6 CC2D2A Meckel syndrome 13 CC2D2A Hennekam syndrome 1 CCBE1 Primary ciliary dyskinesia 21 CCDC103 Situs ambiguus 6 CCDC11 Situs inversus totalis 3 CCDC11 Primary ciliary dyskinesia 21 CCDC114 Primary ciliary dyskinesia 21 CCDC39 Primary ciliary dyskinesia 21 CCDC40

Table IX-21781338.1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 CCDC50 Papillary or follicular thyroid carcinoma 14 CCDC6 Congenital myopathy with internal nuclei and atypical cores 1 CCDC78 3M syndrome 3 CCDC8 Congenital non-communicating hydrocephalus 1 CCDC88C Hereditary cerebral cavernous malformation 3 CCM2 B-cell chronic lymphocytic leukemia 7 CCND1 Mantle cell lymphoma 3 CCND1 Multiple myeloma 1 CCND1 Hereditary sensory and autonomic neuropathy with spastic paraplegia 1 CCT5 Fetal and neonatal alloimmune thrombocytopenia 6 CD109 Nephrotic syndrome-deafness-pretibial epidermolysis bullosa syndrome 1 CD151 Common variable immunodeficiency 10 CD19 Juvenile rheumatoid factor-negative polyarthritis 9 CD247 Oligoarticular juvenile arthritis 9 CD247 T-B+ severe combined immunodeficiency due t o CD3delta/CD3epsilon/CD3zeta 3 CD247 Autosomal recessive lymphoproliferative disease 2 CD27 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 CD2AP Methylmalonic aciduria due to transcobalamin receptor defect 1 CD320 T-B+ severe combined immunodeficiency due to CD3delta/CD3epsilon/CD3zeta 3 CD3D T-B+ severe combined immunodeficiency due to CD3delta/CD3epsilon/CD3zeta 3 CD3E Combined immunodeficiency due to CD3gamma deficiency 1 CD3G Hyper-lgM syndrome type 3 1 CD40 X-linked hyper-lgM syndrome 1 CD40LG Atypical hemolytic uremic syndrome with MCP/CD46 anomaly 1 CD46 Primary CD59 deficiency 1 CD59 Autosomal agammaglobulinemia 7 CD79A Autosomal agammaglobulinemia 7 CD79B Common variable immunodeficiency 10 CD81 Susceptibility t o respiratory infections associated with CD8alpha chain mutation 1 CD8A C syndrome 1 CD96 Ear-patella-short stature syndrome 5 CDC6 Familial isolated hyperparathyroidism 3 CDC73 Familial parathyroid adenoma 2 CDC73 Hyperparathyroidism - jaw tumor syndrome 1 CDC73 Parathyroid carcinoma 2 CDC73 Familial gastric cancer 2 CDH 1

Table IX-21781338.1 Gastric linitis plastica 1 CDH1 Autosomal dominant nonsyndromic intellectual deficit 15 CDH15 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 CDH23 Usher syndrome type 1 9 CDH23 EEM syndrome 1 CDH3 Hypotrichosis with juvenile macular degeneration 1 CDH3 Cone rod dystrophy 22 CDHR1 Retinitis pigmentosa 61 CDHR1 Dedifferentiated liposarcoma 3 CDK4 Familial melanoma 6 CDK4 Well-differentiated liposarcoma 3 CDK4 Autosomal recessive primary microcephaly 11 CDK5RAP2 Atypical Rett syndrome 4 CDKL5 West syndrome 4 CDKL5 Multiple endocrine neoplasia type 1 5 CDKN1A Multiple endocrine neoplasia type 1 5 CDKN 1B Multiple endocrine neoplasia type 4 1 CDKN1B Beckwith-Wiedemann syndrome due t o CDKN1C mutation 1 CDKN1C IMAGe syndrome 1 CDKN1C Familial melanoma 6 CDKN2A Familial pancreatic carcinoma 8 CDKN2A Melanoma neural system tumor syndrome 1 CDKN2A Melanoma-pancreatic cancer syndrome 1 CDKN2A Precursor B-cell acute lymphoblastic leukemia 13 CDKN2A Precursor T-cell acute lymphoblastic leukemia 19 CDKN2A Familial melanoma 6 CDKN2B Multiple endocrine neoplasia type 1 5 CDKN2B Multiple endocrine neoplasia type 1 5 CDKN2C Familial melanoma 6 CDKN2D Alobar holoprosencephaly 14 CDON Lobar holoprosencephaly 14 CDON Microform holoprosencephaly 14 CDON Midline interhemispheric variant of holoprosencephaly 14 CDON Semilobar holoprosencephaly 14 CDON Septopreoptic holoprosencephaly 14 CDON Generalized peeling skin syndrome type B 1 CDSN Hypotrichosis simplex of the scalp 2 CDSN Ear-patella-short stature syndrome 5 CDT1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 CEACAM16 Acute myeloid leukemia with CEBPA somatic mutations 1 CEBPA Acute myeloid leukemia with t(8;21)(q22;q22) translocation 3 CEBPA

Table IX-21781338.1 Inherited acute myeloid leukemia 1 CEBPA Recurrent infection due t o specific granule deficiency 1 CEBPE Cat-eye syndrome 2 CECR1 Cat-eye syndrome 2 CECR2 MODY syndrome 12 CEL Autosomal recessive primary microcephaly 11 CENPJ Seckel syndrome 6 CENPJ Autosomal recessive primary microcephaly 11 CEP152 Seckel syndrome 6 CEP152 Senior-Loken syndrome 8 CEP164 Bardet-Biedl syndrome 17 CEP290 Joubert syndrome with oculorenal defect 6 CEP290 Leber congenital amaurosis 18 CEP290 Meckel syndrome 13 CEP290 Senior-Loken syndrome 8 CEP290 Joubert syndrome 9 CEP41 Joubert syndrome with ocular defect 5 CEP41 Mosaic variegated aneuploidy syndrome 2 CEP57 Autosomal recessive primary microcephaly 11 CEP63 Isolated cytochrome C oxidase deficiency 9 CEP89 Retinitis pigmentosa 6 1 CERKL Cholesterol-ester transfer protein deficiency 2 CETP Atypical hemolytic uremic syndrome with B factor anomaly 1 CFB Biliary atresia with splenic malformation syndrome 1 CFC1 Double outlet right ventricle 3 CFC1 Situs ambiguus 6 CFC1 Recurrent Neisseria infections due t o factor D deficiency 1 CFD Dense deposit disease 2 CFH Familial drusen 2 CFH Immunodeficiency with factor H anomaly 1 CFH Immunoglobulin-mediated membranoproliferative glomerulonephritis 2 CFH Atypical hemolytic uremic syndrome with anti-factor H antibodies 3 CFHR1 C3 glomerulonephritis 2 CFHR1 Dense deposit disease 2 CFHR1 Atypical hemolytic uremic syndrome with anti-factor H antibodies 3 CFHR3 C3 glomerulonephritis 2 CFHR5 Atypical hemolytic uremic syndrome with 1factor anomaly 1 CFI Immunodeficiency with factor 1anomaly 1 CFI Typical nemaline myopathy 4 CFL2 Properdin deficiency 1 CFP Congenital bilateral absence of vas deferens 1 CFTR

7 1

Table IX-21781338.1 Cystic fibrosis 4 CFTR Hereditary chronic pancreatitis 6 CFTR Idiopathic bronchiectasis 4 CFTR CHARGE syndrome 2 CHD7 Kallmann syndrome 19 CHD7 Normosmic congenital hypogonadotropic hypogonadism 18 CHD7 Omenn syndrome 9 CHD7 Familial prostate cancer 14 CHEK2 Hereditary breast and ovarian cancer syndrome 13 CHEK2 Li-Fraumeni syndrome 2 CHEK2 Osteosarcoma, somatic 1 CHEK2 Amyotrophic lateral sclerosis 23 CHGB Congenital muscular dystrophy due t o phosphatidyl choline biosynthesis defect 1 CHKB Distal monosomy 3p 3 CHL1 Choroideremia 2 CHM Pontocerebellar hypoplasia type 8 1 CHMP1A Amyotrophic lateral sclerosis 23 CHMP2B Behavioral variant of frontotemporal dementia 3 CHM P2B Posterior polar cataract 5 CHM P4B Duane syndrome 2 CHN1 Isolated congenital megalocornea 1 CHRDL1 Prune belly syndrome 1 CHRM3 Lethal multiple pterygium syndrome 4 CHRNA1 Postsynaptic congenital myasthenic syndromes 9 CHRNA1 Nocturnal frontal lobe epilepsy 5 CHRNA2 Nocturnal frontal lobe epilepsy 5 CHRNA4 15ql3.3 microdeletion syndrome 1 CHRNA7 Postsynaptic congenital myasthenic syndromes 9 CHRNB1 Nocturnal frontal lobe epilepsy 5 CHRN B2 Lethal multiple pterygium syndrome 4 CHRND Postsynaptic congenital myasthenic syndromes 9 CHRND Postsynaptic congenital myasthenic syndromes 9 CHRNE Autosomal recessive multiple pterygium syndrome 1 CHRNG Lethal multiple pterygium syndrome 4 CHRNG Ehlers-Danlos syndrome, musculocontractural type 2 CHST14 CHST3-related skeletal dysplasia 1 CHST3 Macular corneal dystrophy 1 CHST6 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 5 2 CIB2 Usher syndrome type 1 9 CIB2 Immunodeficiency by defective expression of HLA class 2 5 CIITA

Table IX-21781338.1 Wolfram syndrome 2 CISD2 Atrial septal defect, ostium secundum type 8 CITED2 Atrial septal defect, sinus venosus type 1 CITED2 Single ventricular septal defect 1 CITED2 Situs inversus totalis 3 CITED2 Tetralogy of Fallot 8 CITED2 Ventricular septal defect 4 CITED2 Cystic f ibrosis 4 CLCA4 Cold-induced sweating syndrome 2 CLCF1 Thomsen and Becker disease 1 CLCN1 Juvenile myoclonic epilepsy 7 CLCN2 Dent disease type 1 1 CLCN5 Albers-Schonberg osteopetrosis 1 CLCN7 Autosomal recessive malignant osteopetrosis 5 CLCN7 Intermediate osteopetrosis 2 CLCN7 Infantile Bartter syndrome with deafness 3 CLCNKA Classic Bartter syndrome 1 CLCNKB Gitelman syndrome 2 CLCNK B Gitelman syndrome 2 CLCNKB Infantile Bartter syndrome with deafness 3 CLCNKB Ichthyosis - hypotrichosis - sclerosing cholangitis 1 CLDN1 Autosomal recessive no.nsyndromic sensorineural deafness type DFNB 5 2 CLDN14 Familial hypomagnesemia - hypercalciuria - nephrocalcinosis 1 CLDN16 Familial hypomagnesemia - hypercalciuria - nephrocalcinosis - severe ocular involvement 1 CLDN19 Chronic mucocutaneous candidiasis 6 CLEC7A X-linked intellectual deficit - cardiomegaly - congestive heart failure 1 CLIC2 Williams syndrome 17 CLIP2 Congenital short bowel syndrome 2 CLMP CLN3 disease 1 CLN3 CLN5 disease 1 CLN5 CLN4A disease 1 CLN6 CLN6 disease 1 CLN6 CLN8 disease 1 CLN8 Progressive epilepsy - intellectual deficit, Finnish type 1 CLN8 Perrault syndrome 4 CLP Retinitis pigmentosa 6 1 CLRN 1 Usher syndrome type 3 3 CLRN 1 Inflammatory myofibroblastic tumor 6 CLTC Translocation renal cell carcinoma 7 CLTC Proximal myotonic myopathy 1 CNBP

Table IX-21781338.1 Retinitis pigmentosa 6 1 CNGA1 Achromatopsia 6 CNGA3 Retinitis pigmentosa 6 1 CNGB1 Achromatopsia 6 CNGB3 Progressive cone dystrophy 4 CNGB3 Familial primary hypomagnesemia with normocalcuria and normocalcemia 2 CNNM2 Jalili syndrome 1 CNNM4 Precursor T-cell acute lymphoblastic leukemia 19 CNOT3 Congenital lethal myopathy, Compton-North type 1 CNTN 1 Benign adult familial myoclonic epilepsy 1 CNTN2 Distal monosomy 3p 3 CNTN4 Cortical dysplasia - focal epilepsy syndrome 1 CNTNAP2 Pitt-Hopkins-like syndrome 2 CNTNAP2 Isolated cytochrome C oxidase deficiency 9 COA5 Leigh syndrome with cardiomyopathy 8 COA5 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 COCH Meniere disease 1 COCH COG1-CDG syndrome 1 COG1 COG4-CDG syndrome 1 COG4 COG5-CDG syndrome 1 COG5 COG7-CDG syndrome 1 COG7 COG8-CDG syndrome 1 COG8 Metaphyseal chondrodysplasia, Schmid type 1 COL10A1 Fibrochondrogenesis 2 COLllAl Marshall syndrome 1. COL11A1 Stickler syndrome type 2 1 COLllAl Autosomal dominant nonsyndromic sensorineural deafness type DFNA . 30 COL11A2 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 COL11A2 Fibrochondrogenesis 2 COL11A2 Otospondylomegaepiphyseal dysplasia 2 COL11A2 Stickler syndrome type 3 1 COL11A2 Weissenbacher- Zweymuller syndrome 1 COL11A2 Punctate palmoplantar keratoderma type 2 COL14A1 Generalized junctional epidermolysis bullosa, non-Herlitz type 5 COL17A1 Late-onset junctional epidermolysis bullosa 1 COL17A1 Localized junctional epidermolysis bullosa, non-Herlitz type 2 COL17A1 Caffey disease 1 COL1A1 Dermatofibrosarcoma protuberans 2 COL1A1 Ehlers-Danlos syndrome type 1 3 COL1A1 Ehlers-Danlos syndrome type 7A 1 COL1A1 Ehlers-Danlos syndrome, vascular-like type 1 COL1A1

Table IX-21781338.1 Ehlers-Danlos/osteogenesis imperfecta syndrome 2 COL1A1 High bone mass osteogenesis imperfecta 2 COL1A1 Osteogenesis imperfecta type 1 2 COL1A1 Osteogenesis imperfecta type 2 5 COL1A1 Osteogenesis imperfecta type 3 9 COL1A1 Osteogenesis imperfecta type 4 7 COL1A1 Eh!ers-Danlos syndrome type 7B 1 COL1A2 Ehlers-Danlos syndrome, cardiac valvular type 1 COL1A2 Ehlers-Danlos/osteogenesis imperfecta syndrome 2 COL1A2 High bone mass osteogenesis imperfecta 2 COL1A2 Osteogenesis imperfecta type 1 2 COL1A2 Osteogenesis imperfecta type 2 5 COL1A2 Osteogenesis imperfecta type 3 9 COL1A2 Osteogenesis imperfecta type 4 7 COL1A2 Achondrogenesis type 2 1 COL2A1 Autosomal dominant rhegmatogenous retinal detachment 1 COL2A1 Czech dysplasia, metatarsal type 1 COL2A1 Familial avascular necrosis of femoral head 1 COL2A1 Hypochondrogenesis 1 COL2A1 Kniest dysplasia 1 COL2A1 Legg-Calve-Perthes disease 1 COL2A1 Mild spondyloepiphyseal dysplasia due to COL2A1 mutation with early- onset osteoarthritis 1 COL2A1 Multiple epiphyseal dysplasia, Beighton type 1 COL2A1 Otospondylomegaepiphyseal dysplasia 2 COL2A1 Platyspondylic dysplasia, Torrance type 1 COL2A1 Spondyloepimetaphyseal dysplasia congenita, Strudwick type 1 COL2A1 Spondyloepiphyseal dysplasia congenita 1 COL2A1 Spondylometaphyseal dysplasia, 'corner fracture' type 1 COL2A1 Spondyloperipheral dysplasia - short ulna 1 COL2A1 Stickler syndrome type 1 1 COL2A1 Acrogeria 1 COL3A1 Ehlers-Danlos syndrome, vascular type 1 COL3A1 Familial abdominal aortic aneurysm 1 COL3A1 Autosomal dominant familial hematuria - retinal arteriolar tortuosity - contractures 1 COL4A1 Familial vascular leukoencephalopathy 1 COL4A1 Schizencephaly 4 COL4A1 Walker-Warburg syndrome 13 COL4A1 Familial porencephaly 2 COL4A2 Autosomal dominant Alport syndrome 2 COL4A3

Table IX-21781338.1 Autosomal recessive Alport syndrome 2 COL4A4 X-linked Alport syndrome 1 COL4A5 X-linked diffuse leiomyomatosis - Alport syndrome 2 COL4A5 X-linked diffuse leiomyomatosis - Alport syndrome 2 COL4A6 Ehlers-Danlos syndrome type 1 3 COL5A1 Ehlers-Danlos syndrome type 2 2 COL5A1 Ehlers-Danlos syndrome type 1 3 COL5A2 Ehlers-Danlos syndrome type 2 2 COL5A2 Bethlem myopathy 3 COL6A1 Congenital muscular dystrophy, Ullrich type 3 COL6A1 Bethlem myopathy 3 COL6A2 Congenital muscular dystrophy, Ullrich type 3 COL6A2 Myosclerosis 1 COL6A2 Bethlem myopathy 3 COL6A3 Congenital muscular dystrophy, Ullrich type 3 COL6A3 Acral dystrophic epidermolysis bullosa 1 COL7A1 Centripetalis recessive dystrophic epidermolysis bullosa 1 COL7A1 Dystrophic epidermolysis bullosa pruriginosa 1 COL7A1 Dystrophic epidermolysis bullosa, nails only 1 COL7A1 Epidermolysis bullosa simplex superficialis 1 COL7A1 Generalized dominant dystrophic epidermolysis bullosa 1 COL7A1 Pretibial dystrophic epidermolysis bullosa 1 COL7A1 Recessive dystrophic epidermolysis bullosa inversa 1 COL7A1 Recessive dystrophic epidermolysis bullosa-generalized other 1 COL7A1 Severe generalized recessive dystrophic epidermolysis bullosa 2 COL7A1 Transient bullous dermolysis of the newborn 1 COL7A1 Fuchs endothelial corneal dystrophy 4 COL8A2 Posterior polymorphous corneal dystrophy 3 COL8A2 Autosomal recessive Stickler syndrome 2 COL9A1 Multiple epiphyseal dysplasia due t o collagen 9 anomaly 3 COL9A1 Autosomal recessive Stickler syndrome 2 COL9A2 Multiple epiphyseal dysplasia due t o collagen 9 anomaly 3 COL9A2 Multiple epiphyseal dysplasia due t o collagen 9 anomaly 3 COL9A3 Craniofacial-ulnar-renal syndrome 2 COLEC11 Synaptic congenital myasthenic syndromes 2 COLQ Multiple epiphyseal dysplasia type 1 1 COMP Pseudoachondroplasia 1 COMP 22qll.2 deletion syndrome 7 COMT Leigh syndrome with nephrotic syndrome 4 COQ2 Familial steroid-resistant nephrotic syndrome with sensorineural deafness 1 COQ6 Encephalopathy - hypertrophic cardiomyopathy - renal tubular disease 1 COQ9

Table IX-21781338.1 Severe combined immunodeficiency due to COROIA deficiency 1 COROIA Fatal infantile cytochrome C oxidase deficiency 5 COX10 Leigh syndrome with cardiomyopathy 8 COX10 Leigh syndrome with leukodystrophy 13 COX10 Isolated cytochrome C oxidase deficiency 9 COX14 Fatal infantile cytochrome C oxidase deficiency 5 COX15 Leigh syndrome with cardiomyopathy 8 COX15 Isolated cytochrome C oxidase deficiency 9 COX20 Pancreatic insufficiency - anemia - hyperostosis 1 COX4I2 Isolated cytochrome C oxidase deficiency 9 COX6B1 Leigh syndrome with cardiomyopathy 8 COX6B1 Microphthalmia with linear skin defects syndromes 2 COX7B Aceruloplasminemia 1 CP Duane syndrome 2 CPA6 Familial mesial temporal lobe epilepsy with febrile seizures 1 CPA6 Hereditary coproporphyria 1 CPOX Carbamoylphosphate synthetase deficiency 1 CPS1 Carnitine palmitoyi transferase 1A deficiency 1 CPT1A Acute necrotizing encephalopathy of childhood 2 CPT2

Carnitine palmitoyi transferase II deficiency, myopathic form 1 CPT2 Carnitine palmitoyi transferase II deficiency, neonatal form 1 CPT2 Carnitine palmitoyi transferase II deficiency, severe infantile form 1 CPT2 Common variable immunodeficiency 10 CR2 Autosomal recessive nonsyndromic intellectual deficit 15 CRADD Leber congenital amaurosis 18 CRB1 Pigmented paravenous retinochoroidal atrophy 1 CRB1 Retinitis pigmentosa 61 CRB1 Autosomal recessive nonsyndromic intellectual deficit 15 CRBN Distal monosomy 3p 3 CRBN Melanoma of soft part 3 CREB1 Myxofibrosarcoma 3 CREB3L1 Hyperlipoproteinemia type 5 4 CREB3L3 Rubinstein-Taybi syndrome due to CREBBP mutations 1 CREBBP Complete atrioventricular canal 3 CRELD1 Partial atrioventricular canal 3 CRELD1 Distal 22qll.2 microdeletion syndrome 3 CRKL Crisponi syndrome 1 CRLF1 Osteogenesis imperfecta type 2 5 CRTAP Osteogenesis imperfecta type 3 9 CRTAP Osteogenesis imperfecta type 4 7 CRTAP Cone rod dystrophy 22 CRX

Table IX-21781338.1 Leber congenital amaurosis 18 CRX Retinitis pigmentosa 6 1 CRX Cataract-microcomea syndrome 8 CRYAA Nuclear cataract 9 CRYAA Zonular cataract 9 CRYAA Alpha-crystallinopathy 1 CRYAB Familial isolated dilated cardiomyopathy 38 CRYAB Fatal infantile hypertonic myofibrillar myopathy 1 CRYAB Posterior polar cataract 5 CRYAB Zonular cataract 9 CRYAB Cataract with Y-shaped suture opacities 3 CRYBA1 Zonular cataract 9 CRYBA1 Cataract-microcornea syndrome 8 CRYBA4 Microphthalmia - cataract 4 CRYBA4 Zonular cataract 9 CRYBA4 Cataract-microcornea syndrome 8 CRYBB1 Nuclear cataract 9 CRYBB1 Pulverulent cataract 7 CRYBB1 Cataract with Y-shaped suture opacities 3 CRYBB2 Cataract, Coppock-like 4 CRYBB2 Cataract-microcornea syndrome 8 CRYBB2 Cerulean cataract . 4 CRYBB2 Nuclear cataract 9 CRYBB2 Total congenital cataract 5 CRYBB2 Nuclear cataract 9 CRYBB3 Anterior polar cataract 2 CRYGB Total congenital cataract 5 CRYGB Zonular cataract 9 CRYGB Cataract, Coppock-like 4 CRYGC Cataract-microcornea syndrome 8 CRYGC Pulverulent cataract 7 CRYGC Zonular cataract 9 CRYGC Cataract, Coppock-like 4 CRYGD Cataract-microcornea syndrome 8 CRYGD Cerulean cataract 4 CRYGD Coralliform cataract 1 CRYGD Nuclear cataract 9 CRYGD Zonular cataract 9 CRYGD Autosomal dominant childhood-onset cortical cataract 1 CRYGS Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 CRYM Adult-onset leukoencephalopathy with axonal spheroids and pigmented 1 CSF1R

Table IX-21781338.1 glia Congenital pulmonary alveolar proteinosis 5 CSF2RA Congenital pulmonary alveolar proteinosis 5 CSF2RB Atypical chronic myeloid leukemia 1 CSF3R Chronic neutrophilic leukemia 1 CSF3R Hereditary neutrophilia 1 CSF3R Familial advanced sleep-phase syndrome 2 CSNK1D Familial isolated dilated cardiomyopathy 38 CSRP3 Hereditary cerebral hemorrhage with amyloidosis, Icelandic type 1 CST3 Exfoliative ichthyosis 1 CSTA Unverricht-Lundborg disease 3 CSTB Anterior polar cataract 2 CTAA1 Coats plus syndrome 1 CTC1 Dyskeratosis congenita 10 CTC1 Congenital cataracts - facial dysmorphism - neuropathy 1 CTDP1 Diffuse cutaneous systemic sclerosis 4 CTGF Limited cutaneous systemic sclerosis 4 CTGF Cystathioninuria 1 CTH Wegener granulomatosis 3 CTLA4 Autosomal dominant nonsyndromic intellectual deficit 15 CTN NB1 Desmoid disease 2 CTN NB1 Hepatocellular carcinoma, childhood-onset 2 CTN NB1 Pilomatrixoma 1 CTNN B1 Monosomy 5p 2 CTN ND2 Hereditary chronic pancreatitis 6 CTRC Galactosialidosis 1 CTSA Haim-Munk syndrome 1 CTSC Papillon-Lefevre syndrome 1 CTSC CL 10 disease 1 CTSD CLN13 disease 1 CTSF Pycnodysostosis 1 CTSK Grasbeck-lmerslund disease 2 CUBN Pseudohypoaldosteronism type 2 E 1 CU L3 Cabezas syndrome 1 CUL4B 3 M syndrome 3 CUL7 WHIM syndrome 1 CXCR4 46,XY disorder of sex development due t o isolated 17, 20 lyase deficiency 4 CYB5A Recessive hereditary methemoglobinemia type 1 1 CYB5R3 Recessive hereditary methemoglobinemia type 2 1 CYB5R3 ' X-linked mendelian susceptibility to mycobacterial diseases due t o CYBB deficiency . 1 CYBB

Table IX-21781338.1 Autosomal thrombocytopenia with normal platelets 3 CYCS Angelman syndrome 6 CYFI P1 Familial cylindromatosis 1 CYLD Familial multiple trichoepithelioma 1 CYLD 46,XY disorder of sex development - adrenal insufficiency due to CYP11A1 deficiency 2 CYP11A1 Inherited isolated adrenal insufficiency due t o CYP11A1 deficiency 1 CYP11A1 Congenital adrenal hyperplasia due t o 11-beta-hydroxylase deficiency 1 CYP11B1 Familial hyperaldosteronism type 1 2 CYP11B1 Familial hyperaldosteronism type 1 2 CYP11B2 Familial hyperaldosteronism type 1 2 CYP11B2 Familial hyperreninemic hypoaldosteronism type 1 1 CYP11B2 46,XY disorder of sex development due to isolated 17, 20 lyase deficiency 4 CYP17A1 Congenital adrenal hyperplasia due t o 17-alpha-hydroxylase deficiency 1 CYP17A1 Aromatase deficiency 1 CYP19A1 Aromatase excess syndrome 1 CYP19A1 Congenital glaucoma 3 CYP1B1 Juvenile glaucoma 2 CYP1B1 Peters anomaly 6 CYP1B1 Classic congenital adrenal hyperplasia due t o 21-hydroxylase deficiency, salt wasting form 1 CYP21A2 Classic congenital adrenal hyperplasia due t o 21-hydroxylase deficiency, simple virilizing form 1 CYP21A2 Nonclassic congenital adrenal hyperplasia due t o 21-hydroxylase deficiency 1 CYP21A2 Autosomal recessive infantile hypercalcemia 1 CYP24A1 Lethal occipital encephalocele-skeletal dysplasia syndrome 1 CYP26B1 Focal facial dermal dysplasia 2 CYP26C1 Cerebrotendinous xanthomatosis 1 CYP27A1 Hypocalcemic vitamin D dependent rickets 2 CYP27B1 Hypocalcemia vitamin D dependent rickets 2 CYP2R1 Autosomal recessive spastic paraplegia type 56 1 CYP2U1 Lamellar ichthyosis 6 CYP4F22 Bietti crystalline dystrophy 1 CYP4V2 Retinitis pigmentosa 6 1 CYP4V2 Hypercholesterolemia due t o cholesterol 7alpha-hydroxylase deficiency . 1 CYP7A1 Autosomal recessive spastic paraplegia type 5A 1 CYP7B1 Congenital bile acid synthesis defect type 3 1 CYP7B1 D-2-hydroxygl utaric aciduria 2 D2HGDH Craniorachischisis 1 DACT1 Occipital encephalocele 1 DACT1 Autosomal recessive limb-girdle muscular dystrophy - dystroglycanopathy 1 DAG1

Table IX-21781338.1 type C7 Amyotrophic lateral sclerosis 23 DAO Leukoencephalopathy with brain stem and spinal cord involvement - lactate elevation 1 DARS2 Partial chromosome Y deletion 8 DAZ1 Partial chromosome Y deletion 8 DAZ2 Partial chromosome Y deletion 8 DAZ3 Partial chromosome Y deletion 8 DAZ4 Dopamine beta-hydroxylase deficiency 1 DBH Classic maple syrup urine disease 4 DBT Intermediate maple syrup urine disease 5 DBT Intermittent maple syrup urine disease 4 DBT Thiamin-responsive maple syrup urine disease 4 DBT Woodhouse-Sakati syndrome 1 DCAF17 Familial congenital mirror movements 2 DCC Omenn syndrome 9 DCLREIC Severe combined immunodeficiency due to DCLREIC deficiency 1 DCLREIC Congenital stromal corneal dystrophy 1 DCN Amyotrophic lateral sclerosis 23 DCTNl Distal hereditary motor neuropathy type 7 2 DCTN1 Perry syndrome 1 DCTNl Cystic fibrosis 4 DCTN4 Lissencephaly type 1 due to doublecortin gene mutation 1 DCX Subcortical band heterotopia 2 DCX Pentosuria 1 DCXR Xeroderma pigmentosum complementation group E 1 DDB2 Aromatic L-amino acid decarboxylase deficiency 1 DDC Autosomal recessive spastic paraplegia type 28 1 DDHD1 Autosomal recessive spastic paraplegia type 54 1 DDHD2 Myxoid/round cell liposarcoma 2 DDIT3 DDOST-CDG syndrome 1 DDOST Spondyloepimetaphyseal dysplasia - short limb - abnormal calcification 1 DDR2 Warsaw breakage syndrome 1 DDX11 Precursor T-cell acute lymphoblastic leukemia 19 DDX3X Partial chromosome Y deletion 8 DDX3Y Autosomal dominant epilepsy with auditory features 2 DEPDC5 Familial focal epilepsy with variable foci 1 DEPDC5 Nocturnal frontal lobe epilepsy 5 DEPDC5 Autosomal dominant limb-girdle muscular dystrophy type E 1 DES Desminopathy 1 DES Familial isolated dilated cardiomyopathy 38 DES

8 1

Table IX-21781338.1 Scapuloperoneal amyotrophy 4 DES Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DFNA5 Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 DFNB31 Usher syndrome type 2 4 DFNB31 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 DFNB59 Congenital chronic diarrhea with protein-losing enteropathy 1 DGAT1 Atypical hemolytic uremic syndrome with DGKE deficiency 1 DGKE Immunoglobulin-mediated membranoproliferative glomerulonephritis 2 DGKE Adult-onset multiple mitochondrial DNA deletion syndrome due to DGUOK deficiency 1 DGUOK Adult-onset multiple mitochondrial DNA deletion syndrome due t o DGUOK deficiency 1 DGUOK Mitochondrial DNA depletion syndrome, hepatocerebral form due t o DGUOK deficiency 1 DGUOK Desmosterolosis . 1 DHCR24 Smith-Lemli-Opitz syndrome 1 DHCR7 Smith-Magenis syndrome 3 DHCR7 Retinitis pigmentosa 6 1 DHDDS Constitutional megaloblastic anemia with severe neurologic disease 1 DHFR 46,XY complete gonadal dysgenesis 8 DHH 46, XY gonadal dysgenesis - motor and sensory neuropathy 1 DHH Postaxial acrofacial dysostosis 1 DHODH 2-aminoadipic 2-oxoadipic aciduria 1 DHTKD1 Autosomal dominant Charcot-Marie-Tooth disease type 2Q 1 DHTKD1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DIABLO Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DIAPH1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DIAPH3 Familial multinodular goiter 1 DICERl Ovarian malignant Sertoli-Leydig cell tumor 1 DICERl Pleuropulmonary blastoma family tumor susceptibility syndrome 1 DICERl Familial renal cell carcinoma 6 DIRCl Familial renal cell carcinoma 6 DIRC2 Familial renal cell carcinoma 6 DIRC3 Nephroblastoma 5 DIS3L2 Perlman syndrome 1 DIS3L2 Alobar holoprosencephaly 14 DISP1 Lobar holoprosencephaly 14 DISP1 Microform holoprosencephaly 14 DISP1 Midline interhemispheric variant of holoprosencephaly 14 DISP1 Semilobar holoprosencephaly 14 DISP1 Septopreoptic holoprosencephaly 14 DISP1 Dyskeratosis congenita 10 DKC1 82

Table IX-21781338.1 Hoyeraal-Hreidarsson syndrome 4 DKC1 Idiopathic juvenile osteoporosis 4 DKK1 Idiopathic juvenile osteoporosis 4 DKK1 Pyruvate dehydrogenase E2 deficiency 1 DLAT Classic maple syrup urine disease 4 DLD Intermediate maple syrup urine disease 5 DLD Intermittent maple syrup urine disease 4 DLD Leigh syndrome with nephrotic syndrome 4 DLD Pyruvate dehydrogenase E3 deficiency 1 DLD Thiamin-responsive maple syrup urine disease 4 DLD X-linked nonsyndromic intellectual deficit 24 DLG3 Maternal uniparental disomy of chromosome 14 3 DLK1 Paternal uniparental disomy of chromosome 14 3 DLK1 Alobar holoprosencephaly 14 DLL1 Lobar holoprosencephaly 14 DLL1 Microform holoprosencephaly 14 DLL1 Midline interhemispheric variant of holoprosencephaly 14 DLL1 Semilobar holoprosencephaly 14 DLL1 Septopreoptic holoprosencephaly 14 DLL1 Autosomal recessive spondylocostal dysostosis 4 DLL3 Hypomaturation-hypoplastic amelogenesis imperfecta with taurodontism 1 DLX3 Tricho-dento-osseous syndrome 1 DLX3 Split hand - split foot - deafness 1 DLX5 Split hand - split foot - deafness 1 DLX5 Becker muscular dystrophy 1 DMD Congenital stationary night blindness 12 DMD Duchenne muscular dystrophy 2 DMD Familial isolated dilated cardiomyopathy 38 DMD Symptomatic form of muscular dystrophy of Duchenne and Becker in female carriers 1 DMD Dimethylglycine dehydrogenase deficiency 1 DMGDH Autosomal recessive hypophosphatemic rickets 2 DMP1 Steinert myotonic dystrophy 1 DMPK 46,XY complete gonadal dysgenesis 8 DMRT1 46,XY complete gonadal dysgenesis 8 DM T2 Mitochondrial DNA deletion syndrome with progressive myopathy 1 DNA2 Primary ciliary dyskinesia 21 DNAAF2 Primary ciliary dyskinesia 21 DNAAF3 Primary ciliary dyskinesia 21 DNAH 11 Primary ciliary dyskinesia 21 DNAH5 Primary ciliary dyskinesia 21 DNAI 1

Table IX-21781338.1 Primary ciliary dyskinesia 21 DNAI2 Young adult-onset distal hereditary motor neuropathy 1 DNAJB2 Autosomal dominant limb-girdle muscular dystrophy type I D 1 DNAJB6 Dilated cardiomyopathy with ataxia 1 DNAJC19 Williams syndrome 17 DNAJC30 CLN4B disease 1 DNAJC5 Juvenile parkinsonism with intellectual deficit due t o DNAJC6 deficiency 1 DNAJC6 Levodopa-unresponsive juvenile parkinsonism 1 DNAJC6 Primary ciliary dyskinesia 21 DNAL1 Autosomal recessive systemic lupus erythematosus 1 DNASE1L3 Pediatric systemic lupus erythematosus 1 DNASE1L3 Lethal encephalopathy due t o mitochondrial and peroxisomal fission defect 1 DNM1L Autosomal dominant centronuclear myopathy 4 DNM2 Autosomal dominant Charcot-Marie-Tooth disease type 2M 1 DNM2 Autosomal dominant Charcot-Marie-Tooth disease type 2M 1 DNM2 Autosomal dominant intermediate Charcot-Marie-Tooth disease type B 1 DNM2 Cerebellar ataxia-deafness-narcolepsy syndrome 1 DNMT1 Hereditary sensory and autonomic neuropathy type 1 4 DNMT1 ICF syndrome 2 DN MT3B Adams-Oliver syndrome 4 DOCK6 Autosomal dominant nonsyndromic intellectual deficit 15 DOCK8 Autosomal recessive hyper IgE syndrome 1 DOCK8 Combined immunodeficiency due t o DOCK8 deficiency 1 DOCK8 Fetal akinesia deformation sequence. 2 DOK7 Postsynaptic congenital myasthenic syndromes 9 DOK7 DK1-CDG syndrome 1 DOLK Familial isolated dilated cardiomyopathy 38 DOLK Congenital myasthenic syndromes with glycosylation defect 4 DPAGT1 DPAGT1-CDG syndrome 1 DPAGT1 Diffuse panbronchiolitis 2 DPC 1 DPM1-CDG syndrome 1 DPMI DPM2-CDG syndrome 1 DPM2 DPM3-CDG syndrome 1 DPM3 Idiopathic ventricular fibrillation, not Brugada type 2 DPP6 Idiopathic pulmonary fibrosis 11 DPP9 Globozoospermia 2 DPY19L2 Dihydropyrimidine dehydrogenase deficiency 1 DPYD Dihydropyrimidinuria 1 DPYS Primary ciliary dyskinesia 21 DRC1 Myoclonic dystonia 11 3 DRD2

Table IX-21781338.1 Benign essential blepharospasm 1 D D5 Cervical dystonia 2 DRD5 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form ' 10 DSC2 Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 DSC2 Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 DSC2 Hereditary hypotrichosis with recurrent skin vesicles 1 DSC3 Ehlers-Danlos syndrome, musculocontractural type 2 DSE Keratosis palmoplantaris striata 3 DSG1 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 DSG2 Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 DSG2 Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 DSG2 Familial isolated dilated cardiomyopathy 38 DSG2 Hypotrichosis simplex 6 DSG4 Monilethrix 4 DSG4 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 DSP Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 DSP Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 DSP Idiopathic pulmonary fibrosis 11 DSP Keratosis palmoplantaris striata 3 DSP Lethal acantholytic epidermolysis bullosa 2 DSP Skin fragility-woolly hair-palmoplantar keratoderma syndrome 1 DSP Woolly hair-palmoplantar keratoderma-dilated cardiomyopathy syndrome 1 DSP Dentin dysplasia type 1 1 DSPP Dentin dysplasia type II 1 DSPP Dentinogenesis imperfecta type 2 1 DSPP Dentinogenesis imperfecta type 3 1 DSPP Autosomal recessive epidermolysis bullosa simplex 2 DST Hereditary sensory and autonomic neuropathy type 6 1 DST Left ventricular noncompactioh 11 DTNA Hermansky-Pudlak syndrome type 7 1 DTNBP1 Familial thyroid dyshormonogenesis 6 DUOX2 Genetic transient congenital hypothyroidism 1 DUOX2 Familial thyroid dyshormonogenesis 6 DUOXA2 Kallmann syndrome 19 DUSP6 Normosmic congenital hypogonadotropic hypogonadism 18 DUSP6 Facioscapulohumeral dystrophy 4 DUX4 Dyggve-Melchior-Clausen disease 1 DYM Smith-McCort dysplasia 2 DYM Autosomal dominant Charcot-Marie-Tooth disease type 20 1 DYNC1H1 85

Table IX-21781338.1 Autosomal dominant nonsyndromic intellectual deficit 15 DYNC1H1 Childhood-onset proximal spinal muscular atrophy, autosomal dominant 1 DYNC1H1 Jeune syndrome 6 DYNC2H1 Short rib-polydactyly syndrome, Majewski type 2 DYNC2H1 Short rib-polydactyly syndrome, Verma-Naumoff type 3 DYNC2H1 Autosomal dominant nonsyndromic intellectual deficit 15 DYRK1A Autosomal recessive limb-girdle muscular dystrophy type 2B 1 DYSF Congenital myopathy, Paradas type 1 DYSF Distal myopathy with anterior tibial onset 1 DYSF Miyoshi myopathy 2 DYSF Primary dystonia, DYT13 type 1 DYT13 Myoclonic dystonia 15 1 DYT15 Primary dystonia, DYT2 type 2 DYT17 Primary dystonia, DYT2 type 2 DYT2 Primary dystonia, DYT21 type 1 DYT21 Autosomal dominant focal dystonia, DYT7 type 1 DYT7 Leukoencephalopathy - thalamus and brainstem anomalies - high lactate 1 EARS2 X-linked dominant chondrodysplasia punctata 1 EBP Hirschsprung disease 7 ECE1 Distal arthrogryposis type 5D 1 ECEL1 Lipoid proteinosis 1 ECM1 Oligodontia 10 EDA X-linked hypohidrotic ectodermal dysplasia 2 EDA X-linked hypohidrotic ectodermal dysplasia 2 EDA2R Autosomal dominant hypohidrotic ectodermal dysplasia 3 EDAR Autosomal recessive hypohidrotic ectodermal dysplasia 3 EDAR Autosomal dominant hypohidrotic ectodermal dysplasia 3 EDARADD Autosomal recessive hypohidrotic ectodermal dysplasia 3 EDARADD Oligodontia 10 EDARADD Hirschsprung disease 7 EDN3 Ondine syndrome 5 EDN3 Waardenburg-Shah syndrome 3 EDN3 Hirschsprung disease 7 EDNRB Waardenburg-Shah syndrome 3 EDNRB Familial drusen 2 EFEMP1 Autosomal recessive cutis laxa type 1 2 EFEMP2 Lethal arteriopathy syndrome due t o FBLN4 deficiency 1 EFEMP2 Juvenile absence epilepsy 1 EFHC1 Juvenile myoclonic epilepsy 7 EFHC1 Isolated plagiocephaly 3 EFNA4 Craniofrontonasal dysplasia 1 EFNB1

Table IX-21781338.1 Mandibulofacial dysostosis-microcephaly syndrome 1 EFTUD2 Adult hepatocellular carcinoma 1 EGF Familial primary hypomagnesemia with normocalcuria and normocalcemia 2 EGF Giant cell glioblastoma 10 EGFR Gliosarcoma 10 EGFR Autosomal dominant secondary polycythemia 2 EGLN1 Charcot-Marie-Tooth disease type I D 1 EGR2 Charcot-Marie-Tooth disease type 4 E 1 EGR2 Dejerine-Sottas syndrome 4 EGR2 Bifunctional enzyme deficiency 2 EHHADH Kleefstra syndrome due t o 9q34 microdeletion 1 EHMT1 Kleefstra syndrome due to a point mutation 1 EHMT1 Wolcott-Rallison syndrome 1 EIF2AK3 Cree leukoencephalopathy 5 EIF2B1 Ovarioleukodystrophy 5 EIF2B1 Cree leukoencephalopathy 5 EIF2B2 Ovarioleukodystrophy . 5 EIF2B2 Cree leukoencephalopathy 5 EIF2B3 Ovarioleukodystrophy 5 EIF2B3 Cree leukoencephalopathy 5 EIF2B4 Ovarioleukodystrophy 5 EIF2B4 Cree leukoencephalopathy 5 EIF2B5 Ovarioleukodystrophy 5 EIF2B5 Young adult-onset Parkinsonism 13 EIF4G1 Williams syndrome 17 EIF4H Familial prostate cancer 14 ELAC2 Autosomal dominant severe congenital neutropenia 2 ELANE Cyclic neutropenia 1 ELAN E Short stature due t o isolated growth hormone deficiency with X-linked hypogammaglobulinemia 2 ELF4 Autosomal dominant cutis laxa 2 ELN Supravalvular aortic stenosis 1 ELN Williams syndrome 17 ELN Congenital ichthyosis - intellectual deficit - spastic quadriplegia 1 ELOVL4 Stargardt disease 3 ELOVL4 X-linked Emery-Dreifuss muscular dystrophy 2 EMD Bowen-Conradi syndrome 1 EMG1 Schizencephaly 4 EMX2 Hypoplastic amelogenesis imperfecta 1 ENAM Generalized juvenile polyposis/juvenile polyposis coli 3 ENG Rendu-Osler-Weber disease 3 ENG

Table IX-21781338.1 Glycogen storage disease due t o muscle beta-enolase deficiency 1 EN03 Autosomal recessive hypophosphatemic rickets 2 ENPP1 Generalized arterial calcification of infancy 2 ENPP1 Adams-Oliver syndrome 4 EOGT Microcephaly - polymicrogyria - corpus callosum agenesis 1 EOMES Rubinstein-Taybi syndrome due t o EP300 haploinsufficiency 1 EP300 Autosomal dominant secondary polycythemia 2 EPAS1 Multiple paragangliomas associated with polycythemia 1 EPAS1 Sporadic pheochromocytoma 1 EPAS1 Sporadic secreting paraganglioma 1 EPAS1 Hereditary elliptocytosis 3 EPB41 Autosomal dominant nonsyndromic intellectual deficit 1 EPB41L1 Hereditary spherocytosis 5 EPB42 Hereditary nonpolyposis colon cancer 11 EPCAM Intestinal epithelial dysplasia 1 EPCAM Vici syndrome 1 EPG5 Posterior polar cataract 5 EPHA2 Total congenital cataract 5 EPHA2 Familial prostate cancer 14 EPHB2 Familial hypercholanemia 3 EPHX1 Fetal hydantoin syndrome 1 EPHX1 Lafora disease 2 EPM2A Primary familial polycythemia 1 EPOR Primary familial polycythemia 1 EPOR Marie Unna hereditary hypotrichosis 2 EPS8L3 Lethal congenital contracture syndrome type 2 1 ERBB3 Distal monosomy 12p 1 ERC1 Papillary or follicular thyroid carcinoma 14 ERC1 Cockayne syndrome type 2 3 ERCC1 COFS syndrome 4 ERCC1 Beta-thalassemia - trichothiodystrophy 1 ERCC2 COFS syndrome 4 ERCC2 IBI DS syndrome 3 ERCC2 PIBIDS syndrome 3 ERCC2 Xeroderma pigmentosum complementation group D 1 ERCC2 Xeroderma pigmentosum/Cockayne syndrome complex 3 ERCC2 IBIDS syndrome 3 ERCC3 PIBIDS syndrome 3 ERCC3 Xeroderma pigmentosum complementation group B 1 ERCC3 Xeroderma pigmentosum/Cockayne syndrome complex 3 ERCC3 Cockayne syndrome type 1 3 ERCC4

Table IX-21781338.1 Fanconi anemia 16 ERCC4 Xeroderma pigmentosum complementation group F 1 ERCC4 COFS syndrome 4 ERCC5 Xeroderma pigmentosum complementation group G 1 ERCC5 Xeroderma pigmentosum/Cockayne syndrome complex 3 ERCC5 . Cockayne syndrome type 1 3 ERCC6 Cockayne syndrome type ' 2 3 ERCC6 Cockayne syndrome type 3 2 ERCC6 COFS syndrome 4 ERCC6 UV-sensitive syndrome 3 ERCC6 Cockayne syndrome type 1 3 ERCC8 Cockayne syndrome type 2 3 ERCC8 Cockayne syndrome type 3 2 ERCC8 UV-sensitive syndrome 3 ERCC8 Crouzon disease 2 ERF Isolated cloverleaf skull syndrome 2 ERF Isolated scaphocephaly 3 ERF Ewing sarcoma 5 ERG Autosomal recessive spastic paraplegia type 18 1 ERLI N2 Juvenile primary lateral sclerosis 2 ERLIN2 Recessive intellectual disability - motor dysfunction - multiple joint contractures 1 ERLIN2 Roberts syndrome 1 ESC02 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 5 2 ESPN Estrogen resistance syndrome 1 ESR1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 5 2 ESRRB Glutaric acidemia type 2 3 ETFA Glutaric acidemia type 2 3 ETFB Glutaric acidemia type 2 3 ETFDH Ethylmalonic encephalopathy 1 ETHE1 Ewing sarcoma 5 ETVl Ewing sarcoma 5 ETV4 Chronic myelomonocytic leukemia 2 ETV6 Congenital mesoblastic nephroma 2 ETV6 Fibrosarcoma 2 ETV6 Precursor B-cell acute lymphoblastic leukemia 13 ETV6 Acrofacial dysostosis, Weyers type 2 EVC Ellis Van Creveld syndrome 2 EVC Acrofacial dysostosis, Weyers type 2 EVC2 Ellis Van Creveld syndrome 2 EVC2 Desmoplastic small round cell tumor 2 EWSR1

Table IX-21781338.1 Ewing sarcoma 5 EWSR1 Melanoma of soft part 3 EWSR1 Pontocerebellar hypoplasia type 1 < 4 EXOSC3 Chondrosarcoma 1 EXT1 Langer-Giedion syndrome 2 EXT1 Multiple osteochondromas 2 EXT1 Multiple osteochondromas 2 EXT2 Potocki-Shaffer syndrome 3 EXT2 BO syndrome 3 EYAl Branchio-otic syndrome 2 EYA1 Otofaciocervical syndrome 1 EYAl Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 EYA4 Sensorineural deafness with dilated cardiomyopathy 1 EYA4 Retinitis pigmentosa 6 1 EYS Weaver syndrome 2 EZH2 Congenital factor X deficiency 1 F10 Congenital factor XI deficiency 1 Fll Congenital factor XII deficiency 1 F12 Hereditary angioedema type 3 1 F12 Congenital factor XII I deficiency 2 F13A1 Congenital factor XII I deficiency 2 F13B

Congenital factor II deficiency 1 F2 Budd-Chiari syndrome 2 F5 Congenital factor V deficiency 1 F5 Congenital factor VII deficiency 1 F7 Mild hemophilia A 1 F8 Moderately severe hemophilia A 1 F8 Severe hemophilia A 1 F8 Symptomatic form of hemophilia A in female carriers 1 F8 Mild hemophilia B 1 F9 Moderately severe hemophilia B 1 F9 Severe hemophilia B 1 F9 Symptomatic form of hemophilia B in female carriers 1 F9 Autosomal recessive spastic paraplegia type 35 1 FA2H Fatty acid hydroxylase-associated neurodegeneration 1 FA2H FADD-related immunodeficiency 1 FADD Oculootodental syndrome 2 FADD Tyrosinemia type 1 1 FAH Autosomal dominant Kenny-Caffey syndrome 1 FAM111A Osteocraniostenosis 1 FAM111A Hypomyelination - congenital cataract . 1 FAM126A

Table IX-21781338.1 Hereditary sensory and autonomic neuropathy type 2 4 FAM134B Idiopathic pulmonary fibrosis 11 FAM13A Retinitis pigmentosa 6 1 FAM161A Amelogenesis imperfecta - nephrocalcinosis 1 FAM20A Amelogenesis imperfecta and gingival hyperplasia syndrome 1 FAM20A Lethal osteosclerotic bone dysplasia 1 FAM20C Syndactyly - telecanthus - anogenital and renal malformations 1 FAM58A Hypocalcified amelogenesis imperfecta 2 FAM83H Late-onset autosomal recessive medullary cystic kidney disease 3 FAN1 Fanconi anemia 16 - FANCA Fanconi anemia 16 FANCB VACTERL with hydrocephalus 1 FANCB Fanconi anemia 16 FANCC Fanconi anemia 16 FANCD2 Fanconi anemia 16 FANCE Fanconi anemia 16 FANCF Fanconi anemia 16 FANCG Fanconi anemia 16 FANCI Fanconi anemia 16 FANCL Fanconi anemia 16 FANCM Combined oxidative phosphorylation defect type 14 1 FARS2 Autoimmune lymphoproliferative syndrome 6 FAS Juvenile rheumatoid factor-negative polyarthritis 9 FAS Oligoarticular juvenile arthritis 9 FAS Autoimmune lymphoproliferative syndrome 6 FASLG FASTKD2-related infantile mitochondrial encephalomyopathy 1 FASTKD2 Isolated cytochrome C oxidase deficiency 9 FASTKD2 Synpolydactyly type 2 1 FBLN1 Autosomal dominant cutis laxa 2 FBLN5 Autosomal recessive cutis laxa type 1 2 FBLN5 Hereditary sensorimotor neuropathy with hyperelastic skin 1 FBLN5 Acromicric dysplasia 1 FBN1 Acromicric dysplasia 1 FBN1 Ectopia lentis syndrome 2 FBN1 Familial thoracic aortic aneurysm and aortic dissection 8 FBN1 Geleophysic dysplasia 2 FBN1 Glaucoma - ectopia - microspherophakia - stiff joints - short stature 1 FBN1 Marfan syndrome type 1 1 FBN1 Neonatal Marfan syndrome 1 FBN1 Progeroid and marfanoid aspect-lipodystrophy syndrome 1 FBN1 Shprintzen-Goldberg syndrome 2 FBN 1

9 1

Table IX-21781338.1 Stiff skin syndrome 1 FBN1 Weill-Marchesani syndrome 4 FBN1 Congenital contractural arachnodactyly 1 FBN2 Fructose-l,6-bisphosphatase deficiency 1 FBP1 Parkinsonian-pyramidal syndrome 2 FBX07 Split hand-split foot malformation 4 FBXW4 Immunodeficiency due t o ficolin3 deficiency 1 FCN3 Erythropoietic protoporphyria 2 FECH Hereditary acrokeratotic poikiloderma, Weary type 1 FE M T1 Kindler syndrome 2 FERMT1

Leukocyte adhesion deficiency type II I 1 FERMT3 Familial afibrinogenemia 3 FGA Familial dysfibrinogenemia 3 FGA Familial hypodysfibrinogenemia 3 FGA Familial hypofibrinogenemia 3 FGA Familial renal amyloidosis due t o fibrinogen A alpha-chain variant 1 FGA Familial afibrinogenemia 3 FGB Familial dysfibrinogenemia 3 FGB Familial hypodysfibrinogenemia 3 FGB Familial hypofibrinogenemia 3 FGB Aarskog-Scott syndrome 1 FGD1 Charcot-Marie-Tooth disease type 4 H 1 FGD4 Aplasia of lacrimal and salivary glands 1 FGF10 Lacrimo-auriculo-dento-digital syndrome 3 FGF10 Spinocerebellar ataxia type 27 1 FGF14 Kallmann syndrome 19 FGF17 Normosmic congenital hypogonadotropic hypogonadism 18 FGF17 Autosomal dominant hypophosphatemic rickets 1 FGF23 Autosomal dominant hypophosphatemic rickets 1 FGF23 Hypercalcemic tumoral calcinosis 3 FGF23 Deafness with labyrinthine aplasia, microtia, and microdontia 1 FGF3 Oculootodental syndrome 2 FGF3 Otodental syndrome 1 FGF3 Alobar holoprosencephaly 14 FGF8 Kallmann syndrome 19 FGF8 Lobar holoprosencephaly 14 FGF8 Microform holoprosencephaly 14 FGF8 Midline interhemispheric variant of holoprosencephaly 14 FGF8 Normosmic congenital hypogonadotropic hypogonadism 18 FGF8 Semilobar holoprosencephaly 14 FGF8 Septopreoptic holoprosencephaly 14 FGF8

Table IX-21781338.1 Multiple synostoses syndrome 3 FGF9 Giant cell glioblastoma 10 FGFRl . Gliosarcoma 10 FGFRl Isolated trigonocephaly 2 FGFRl Kallmann syndrome 19 FGFRl Myeloid neoplasm associated with FGFRl rearrangement 1 FGFRl Normosmic congenital hypogonadotropic hypogonadism 18 FGFRl Oligodontia 10 FGFRl Osteoglophonic dwarfism 1 FGFRl Pfeiffer syndrome type 1 2 FGFRl Septo-optic dysplasia 6 FGFRl Antley-Bixler syndrome 1 FGFR2 Apert syndrome 1 FGFR2 Crouzon disease 2 FGFR2 Cutis gyrata - acanthosis nigricans - craniosynostosis 2 FGFR2 Familial scaphocephaly syndrome, McGillivray type 1 FGFR2 FGFR2-related bent bone dysplasia 1 FGFR2 Jackson-Weiss syndrome 2 FGFR2 Lacrimo-auriculo-dento-digital syndrome 3 FGFR2 Pfeiffer syndrome type 1 2 FGFR2 Pfeiffer syndrome type 2 1 FGFR2 Pfeiffer syndrome type 3 1 FGFR2 Saethre-Chotzen syndrome 4 FGFR2 Achondroplasia 1 FGFR3 Camptodactyly - tall stature - scoliosis - hearing loss 1 FGFR3 Crouzon syndrome - acanthosis nigricans 1 FGFR3 Cutis gyrata - acanthosis nigricans - craniosynostosis 2 FGFR3 Giant cell glioblastoma 10 FGFR3 Gliosarcoma 10 FGFR3 Hypochondroplasia 1 FGFR3 Isolated brachycephaly 3 FGFR3 Isolated cloverleaf skull syndrome 2 FGFR3 Isolated plagiocephaly 3 FGFR3 Lacrimo-auriculo-dento-digital syndrome 3 FGFR3 Muenke syndrome 1 FGFR3 Muenke syndrome 1 FGFR3 Saethre-Chotzen syndrome 4 FGFR3 Severe achondroplasia - developmental delay - acanthosis nigricans 1 FGFR3 Thanatophoric dwarfism type 1 1 FGFR3

Thanatophoric dwarfism type II 1 FGFR3 Familial afibrinogenemia 3 FGG

Table IX-21781338.1 Familial dysfibrinogenemia 3 FGG Familial hypodysfibrinogenemia 3 FGG Familial hypofibrinogenemia 3 FGG Familial leiomyomatosis 1 FH Fumaric aciduria 1 FH Familial renal cell carcinoma 6 FH IT Reducing body myopathy 1 FH L1 Scapuloperoneal amyotrophy 4 FH L1 X-linked Emery-Dreifuss muscular dystrophy 2 FH L1 X-linked myopathy with postural muscle atrophy 1 FH L1 Familial isolated dilated cardiomyopathy 38 FH L2 Amyotrophic lateral sclerosis 23 FIG4 Charcot-Marie-Tooth disease type 4 J 1 FIG4 Primary lateral sclerosis 1 FIG4 Yunis-Varon syndrome 1 FIG4 Idiopathic hypereosinophilic syndrome 3 FIP1L1 Bruck syndrome 2 FKBP10 Osteogenesis imperfecta type 5 2 FKBP10 Ehlers-Danlos syndrome, kyphoscoliotic and deafness type 1 FKBP14 Williams syndrome 17 FKBP6

Autosomal recessive limb-girdle muscular dystrophy type 2 1 1 FKRP Congenital muscular dystrophy type 1C 1 FKRP Muscle eye brain disease 7 FKRP Walker-Warburg syndrome 13 FKRP Autosomal recessive limb-girdle muscular dystrophy type 2M 1 FKTN Familial isolated dilated cardiomyopathy 38 FKTN Muscle eye brain disease 7 FKTN Walker-Warburg syndrome 13 FKTN Birt-Hogg-Dube syndrome 1 FLCN Familial spontaneous pneumothorax 1 FLCN Ewing sarcoma 5 FLU Paris-Trousseau thrombocytopenia 1 FLU Smith-Magenis syndrome 3 FLU Chronic intestinal pseudo-obstruction 1 FLNA Congenital short bowel syndrome 2 FLNA Congenital valvular dysplasia 1 FLNA Ehlers-Danlos syndrome with periventricular heterotopia 1 FLNA Frontometaphyseal dysplasia 1 FLNA Osteodysplasty, Melnick-Needles type 1 FLNA Otopalatodigital syndrome type 1 1 FLNA Otopalatodigital syndrome type 2 1 FLNA

Table IX-21781338.1 Periventricular nodular heterotopia 2 FLNA Terminal osseous dysplasia - pigmentary defects 1 FLNA Atelosteogenesis 1 1 FLNB Atelosteogenesis type III 1 FLNB Autosomal dominant Larsen syndrome 1 FLNB Boomerang dysplasia 1 FLNB Synspondylism 1 FLNB Distal myopathy with posterior leg and anterior hand involvement 1 FLNC Muscle filaminopathy 1 FLNC Kallmann syndrome 19 FLRT3 Acute biphenotypic leukemia 2 FLT3 Precursor B-cell acute lymphoblastic leukemia 13 FLT3 Precursor T-cell acute lymphoblastic leukemia 19 FLT3 Milroy disease 2 FLT4 - Posterior column ataxia - retinitis pigmentosa 1 FLVCR1 Fowler syndrome 1 FLVCR2 Trimethylaminuria 1 FM03 Fragile X syndrome 1 FMR1 Fragile X-associated tremor/ataxia syndrome 1 FMR1 Xq27.3q28 duplication syndrome 1 FMR1 FRAXE intellectual deficit 2 FMR3 Fibronectin glomerulopathy 1 FN 1 Neurodegenerative syndrome due t o cerebral folate transport deficiency 1 FOLR1 Axenfeld-Rieger syndrome 2 FOXC1 Axenfeld's anomaly 2 FOXC1 Peters anomaly 6 FOXC1 Rieger's anomaly 2 FOXC1 Lymphedema - distichiasis 1 FOXC2 Yellow nail syndrome 1 FOXC2 Vitiligo-associated autoimmune disease 2 FOXD3 Athyreosis 5 FOXE1 Bamforth syndrome 1 FOXEl Papillary or follicular thyroid carcinoma 14 FOXE1 Thyroid hypoplasia 5 FOXE1 Congenital primary aphakia 1 FOXE3 Familial ocular anterior segment mesenchymal dysgenesis 2 FOXE3 Congenital alveolar capillary dysplasia 1 FOXF1 14qll.2 microduplication syndrome 1 FOXG1 14ql2 microdeletion syndrome 1 FOXG1 Atypical Rett syndrome 4 FOXG1 Alobar holoprosencephaly 14 FOXH1

Table IX-21781338.1 Lobar holoprosencephaly 14 FOXH1 Microform holoprosencephaly 14 FOXH1 Midline interhemispheric variant of holoprosencephaly 14 FOXH1 Semilobar holoprosencephaly 14 FOXH1 Septopreoptic holoprosencephaly 14 FOXH1 Pendred syndrome 3 FOXI1 Blepharophimosis - epicanthus inversus - ptosis due to a point mutation 1 FOXL2 Blepharophimosis - epicanthus inversus - ptosis, due to 3q23 microdeletion 1 FOXL2 Severe T-cell immunodeficiency - congenital alopecia - nail dystrophy 1 FOXN1 Alveolar rhabdomyosarcoma ~ FOXOl MALT lymphoma 4 FOXP1 7q31 microdeletion syndrome 1 FOXP2 Childhood apraxia of speech 1 FOXP2 X-linked immune dysregulation - polyendocrinopathy - enteropathy 1 FOXP3 Isolated NADH-CoQ reductase deficiency 25 FOXRED1 Fraser syndrome 3 FRAS1 BNAR syndrome 1 FREM1 Congenital diaphragmatic hernia 2 FREM1 Isolated trigonocephaly 2 FREM1 Oculotrichoanal syndrome 1 FREM1 Fraser syndrome 3 FREM2 Facioscapulohumeral dystrophy 4 FRG1 Retinitis pigmentosa 61 FSCN2 Isolated follicle stimulating hormone deficiency 1 FSHB 46,XX gonadal dysgenesis 4 FSH R Ovarian hyperstimulation syndrome 1 FSH R Formiminoglutamic aciduria 1 FTCD FTHl-related iron overload 1 FTH1 Genetic hyperferritinemia without iron overload 1 FTL Hereditary hyperferritinemia with congenital cataracts 1 FTL Neuroferritinopathy 1 FTL Lethal polymalformative syndrome, Boissel type 1 FTO X-linked nonsyndromic intellectual deficit 24 FTSJl Fucosidosis 1 FUCA1 Amyotrophic lateral sclerosis 23 FUS Frontotemporal dementia with motor neuron disease 3 FUS Juvenile amyotrophic lateral sclerosis 3 FUS Myxofibrosarcoma 3 FUS Myxoid/round cell liposarcoma 2 FUS Arnold-Chiari malformation type II 1 FUZ

Table IX-21781338.1 Caudal regression sequence 2 FUZ Cervical spina bifida aperta 6 FUZ Cervical spina bifida cystica 6 FUZ Cervicothoracic spina bifida aperta 6 FUZ Cervicothoracic spina bifida cystica 6 FUZ Lumbosacral spina bifida aperta 6 FUZ Lumbosacral spina bifida cystica 6 FUZ Thoracolumbosacral spina bifida aperta 6 FUZ Thoracolumbosacral spina bifida cystica 6 FUZ Total spina bifida aperta 6 FUZ Total spina bifida cystica 6 FUZ Upper thoracic spina bifida aperta 6 FUZ Upper thoracic spina bifida cystica 6 FUZ Friedreich ataxia 1 FXN Autosomal dominant primary hypomagnesemia with hypocalcuria 2 FXYD2 Nuclear cataract 9 FYCOl Familial exudative vitreoretinopathy 5 FZD4 Persistent hyperplastic primary vitreous 3 FZD4 Retinopathy of prematurity 3 FZD4 Autosomal recessive nail dysplasia 1 FZD6 Glycogen storage disease due to glucose-6-phosphatase deficiency type a 1 G6PC Autosomal recessive severe congenital neutropenia due t o G6PC3 deficiency 1 G6PC3 Glycogen storage disease due to acid maltase deficiency, adult onset 1 GAA Glycogen storage disease due to acid maltase deficiency, juvenile onset 1 GAA Childhood absence epilepsy 6 GABRA1 Juvenile myoclonic epilepsy 7 GABRA1 Thyrotoxic periodic paralysis 3 GABRA3 Childhood absence epilepsy 6 GABRB3 lp36 deletion syndrome 4 GABRD Generalized epilepsy with febrile seizures-plus context 6 GABRD Juvenile myoclonic epilepsy 7 GABRD Childhood absence epilepsy 6 GABRG2 Dravet syndrome 4 GABRG2 Generalized epilepsy with febrile seizures-plus context 6 GABRG2 Inherited congenital spastic tetraplegia 2 GAD1 Adult Krabbe disease 1 GALC Infantile Krabbe disease 2 GALC Late-infantile/juvenile Krabbe disease 1 GALC Erythrocyte galactose epimerase deficiency 1 GALE Generalized galactose epimerase deficiency 1 GALE

Table IX-21781338.1 Galactokinase deficiency 1 GALK1 Mucopolysaccharidosis type 4A 1 GALNS Hypercalcemic tumoral calcinosis 3 GALNT3 Classic galactosemia 1 GALT Guanidinoacetate methyltransferase deficiency 1 GAMT Giant axonal neuropathy ' 1 GAN Autosomal dominant Charcot-Marie-Tooth disease type 2D 1 GARS Distal hereditary motor neuropathy type 5 3 GARS Alobar holoprosencephaly 14 GAS1 Lobar holoprosencephaly 14 GAS1 Microform holoprosencephaly 14 GAS1 Midline interhemispheric variant of holoprosencephaly 14 GAS1 Semilobar holoprosencephaly 14 GAS1 Septopreoptic holoprosencephaly 14 GAS1 Acute basophilic leukemia 2 GATA1 Beta-thalassemia - X-linked thrombocytopenia 1 GATA1 Blackfan-Diamond anemia 11 GATA1 Congenital dyserythropoietic anemia with thrombocytopenia 1 GATA1 Congenital erythropoietic porphyria 2 GATA1 Acute myeloid leukemia 1 GATA2 Deafness - lymphedema - leukemia 1 GATA2 Monocytopenia with susceptibility to infections 1 GATA2 Myelodysplastic syndromes 2 GATA2 Hypoparathyroidism - deafness - renal disease 1 GATA3 46,XY partial gonadal dysgenesis 7 GATA4 8p23.1 microdeletion syndrome 1 GATA4 Atrial septal defect, ostium secundum type 8 GATA4 Complete atrioventricular canal 3 GATA4 Familial atrial fibrillation 14 GATA4 Partial atrioventricular canal 3 GATA4 Tetralogy of Fallot 8 GATA4 Ventricular septal defect 4 GATA4 Familial atrial fibrillation 14 GATA5 Ventricular septal defect 4 GATA5 Atrial septal defect, ostium secundum type 8 GATA6 Complete atrioventricular canal 3 GATA6 Familial atrial fibrillation 14 GATA6 Pancreatic hypoplasia - diabetes - congenital heart disease 1 GATA6 Partial atrioventricular canal 3 GATA6 Tetralogy of Fallot 8 GATA6 Familial isolated dilated cardiomyopathy 38 GATAD1

Table IX-21781338.1 Autosomal dominant nonsyndromic intellectual deficit 15 GATAD2B Arginine:glycine amidinotransferase deficiency 1 GATM Fetal Gaucher disease 1 GBA Gaucher disease - ophthalmoplegia - cardiovascular calcification 1 GBA Gaucher disease - ophthalmoplegia - cardiovascular calcification 1 GBA Gaucher disease type 1 2 GBA Gaucher disease type 2 1 GBA Gaucher disease type 3 1 GBA Young adult-onset Parkinsonism 13 GBA Autosomal recessive cerebellar ataxia with late-onset spasticity 1 GBA2 Autosomal recessive cerebellar ataxia with late-onset spasticity 1 GBA2 Autosomal recessive spastic paraplegia type 46 1 GBA2 Adult polyglucosan body disease 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, adult neuromuscular form 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, childhood combined hepatic and myopathic form 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, childhood neuromuscular form 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, congenital neuromuscular form 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, fatal perinatal neuromuscular form 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, non progressive hepatic form 1 GBE1 Glycogen storage disease due to glycogen branching enzyme deficiency, progressive hepatic form 1 GBE1 Glutaryl-CoA dehydrogenase deficiency 1 GCDH Autosomal dominant dopa-responsive dystonia 1 GCH1 GTP cyclohydrolase 1deficiency 1 GCH1 Hyperinsulinism due to glucokinase deficiency 1 GCK MODY syndrome 12 GCK Permanent neonatal diabetes mellitus 5 GCK Gamma-glutamylcysteine synthetase deficiency 1 GCLC Familial isolated hypoparathyroidism due to agenesis of parathyroid gland 1 GCM2 Atypical glycine encephalopathy 3 GCSH Infantile glycine encephalopathy 3 GCSH Neonatal glycine encephalopathy 3 GCSH Autosomal dominant Charcot-Marie-Tooth disease type 2 K 1 GDAP1 Autosomal recessive Charcot-Marie-Tooth disease with hoarseness 1 GDAP1 Autosomal recessive intermediate Charcot-Marie-Tooth disease type A 1 GDAP1 Charcot-Marie-Tooth disease type 2H 1 GDAP1 Charcot-Marie-Tooth disease type 4A 1 GDAP1 99

Table IX-21781338.1 Double outlet right ventricle 3 GDF1 Tetralogy of Fallot 8 GDF1 Colobomatous microphthalmia 8 GDF3 Isolated anophthalmia - microphthalmia 5 GDF3 Isolated Klippel-Feil syndrome 3 GDF3 Acromesomelic dysplasia, Grebe type GDF5 Acromesomelic dysplasia, Hunter-Thomson type 1 GDF5 Angel-shaped phalango-epiphyseal dysplasia 1 GDF5 Brachydactyly type Al 2 GDF5 Brachydactyly type A2 3 GDF5 Brachydactyly type C 2 GDF5 Fibular aplasia - complex brachydactyly 1 GDF5 Multiple synostoses syndrome 3 GDF5 Proximal symphalangism 2 GDF5 Colobomatous microphthalmia 8 GDF6 Isolated Klippel-Feil syndrome 3 GDF6 Leber congenital amaurosis 18 GDF6 X-linked nonsyndromic intellectual deficit 24 GDI1 Hirschsprung disease 7 GDNF of Genes per Gene Disease Name Disease Name Alexander disease 1 GFAP Congenital cataract - progressive muscular hypotonia - hearing loss - developmental delay 1 GFER Autosomal dominant severe congenital neutropenia 2 GFI1 Hepatoencephalopathy due to combined oxidative phosphorylation deficiency type 1 1 GFM 1 Congenital myasthenic syndromes with glycosylation defect 4 GFPT1 Body skin hyperlaxity due to vitamin K-dependent coagulation factor deficiency 1 GGCX Hereditary combined deficiency of vitamin K-dependent clotting factors 2 GGCX Gamma-glutamyl transpeptidase deficiency 1 GGT1 Isolated growth hormone deficiency type IA 1 GH1 Isolated growth hormone deficiency type IB 2 GH1 Isolated growth hormone deficiency type II 1 GH1 Short stature due to growth hormone qualitative anomaly 1 GH1 Laron syndrome 1 GHR Short stature due to partial GH deficiency 1 GHR Isolated growth hormone deficiency type IB 2 GHRHR Short stature due to GHSR deficiency 1 GHSR Congenital intrinsic factor deficiency 1 GIF 100

Table IX-21781338.1 Young adult-onset Parkinsonism 13 GIGYF2 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GIPC3 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJA1 Hypoplastic left heart syndrome 2 GJA1 Oculodentodigital dysplasia 1 GJA1 Syndactyly type 3 1 GJA1 Cataract, Coppock-like 4 GJA3 Nuclear cataract 9 GJA3 Posterior polar cataract 5 GJA3 Pulverulent cataract 7 GJA3 Atrial stand still 2 GJA5 Familial atrial fibrillation 14 GJA5 Tetralogy of Fallot 8 GJA5 Cataract-microcornea syndrome 8 GJA8 Pulverulent cataract 7 GJA8 X-linked Charcot-Marie-Tooth disease type 1 1 GJB1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GJB2 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJB2 Hypotrichosis-deafness syndrome 1 GJB2 Keratoderma hereditarium mutilans 1 GJB2 KID syndrome 2 GJB2 Knuckle pads-leuconychia-sensorineural deafness-palmoplantar hyperkeratosis syndrome 1 GJB2 Palmoplantar keratoderma-deafness syndrome 2 GJB2 Porokeratotic eccrine ostial and dermal duct nevus 1 GJB2 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GJB3 Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 GJB3 Erythrokeratodermia variabilis 2 GJB3 Neuropathy with hearing impairment 1 GJB3 Transgrediens et progrediens palmoplantar keratoderma 1 GJB3 Erythrokeratodermia variabilis 2 GJB4 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GJB6 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJB6 Hidrotic ectodermal dysplasia 1 GJB6 KID syndrome 2 GJB6 Autosomal recessive spastic paraplegia type 44 1 GJC2 Autosomal recessive spastic paraplegia type 44 1 GJC2 Milroy disease 2 GJC2 Pelizaeus-Merzbacher-like due to GJC2 mutation 1 GJC2 Glycerol kinase deficiency, adult form 1 GK Glycerol kinase deficiency, infantile form 1 GK

101

Table IX-21781338.1 Fabry disease 1 GLA GM1 gangliosidosis type 2 1 GLB1 GM1 gangliosidosis type 3 1 GLB1 Mucopolysaccharidosis type 4B 1 GLB1 Atypical glycine encephalopathy 3 GLDC Infantile glycine encephalopathy 3 GLDC Neonatal glycine encephalopathy 3 GLDC Lethal arthrogryposis - anterior horn cell disease 1 GLE1 Lethal congenital contracture syndrome type 1 1 GLE1 Alobar holoprosencephaly 14 GLI2 Combined pituitary hormone deficiencies, genetic forms 5 GLI2 Lobar holoprosencephaly 14 GLI2 Microform holoprosencephaly 14 GLI2 Midline interhemispheric variant of holoprosencephaly 14 GLI2 Semilobar holoprosencephaly 14 GLI2 Septopreoptic holoprosencephaly 14 GLI2 Acrocallosal syndrome 2 GLI3 Greig cephalopolysyndactyly syndrome 1 GLI3 Paliister-Hall syndrome 2 GLI3 Polysyndactyly, bilateral 1 GLI3 Polysyndactyly, unilateral 1 GLI3 Postaxial Polydactyly type A, bilateral 2 GLI3 Postaxial Polydactyly type A, unilateral 1 GLI3 Postaxial Polydactyly type B, bilateral 1 GLI3 Postaxial Polydactyly type B, unilateral 1 GLI3 Acute megakaryoblastic leukemia without Down syndrome 2 GLIS2 Juvenile autosomal recessive medullary cystic kidney disease 4 GLIS2 Neonatal diabetes - congenital hypothyroidism - congenital glaucoma - hepatic fibrosis - polycystic kidneys 1 GLIS3 Glomuvenous malformation 1 GLMN Hereditary hyperekplexia 4 GL A 1 Hereditary hyperekplexia 4 GLRB Autosomal recessive pyridoxine-refractory sideroblastic anemia 2 GLRX5 Hyperinsulinism-hyperammonemia syndrome 1 GLU D1 Congenital brain dysgenesis due to glutamine synthetase deficiency 1 GLUL D-glyceric aciduria 1 GLYCTK GM2-gangliosidosis, AB variant 1 GM2A Auriculo-condylar syndrome 2 GNAI3 Autosomal dominant focal dystonia, DYT25 1 GNAL Nevi flammei 1 GNAQ Sturge-Weber syndrome 1 GNAQ

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Table IX-21781338.1 Fibrous dysplasia of bone 1 GNAS Mazabraud syndrome 1 GNAS McCune-Albright syndrome 1 GNAS Progressive osseous heteroplasia 1 GNAS Pseudohypoparathyroidism type 1A 1 GNAS Pseudohypoparathyroidism type 1A 1 GNAS Pseudohypoparathyroidism type I B 2 GNAS Pseudohypoparathyroidism type 1C 1 GNAS Pseudopseudohypoparathyroidism 1 GNAS Congenital stationary night blindness 12 GNAT1 Achromatopsia 6 GNAT2 Progressive cone dystrophy 4 GNAT2 Autosomal dominant intermediate Charcot-Marie-Tooth disease type F 1 GNB4 Distal myopathy, Nonaka type 1 GNE Distal myopathy, Nonaka type 1 GNE Sialuria 1 GNE Hypermethioninemia due to glycine N-methyltransferase deficiency 1 GNMT Rhizomelic chondrodysplasia punctata type 2 1 GNPAT Mucolipidosis type 2 1 GNPTAB Mucolipidosis type 3 2 GNPTAB Mucolipidosis type 3 2 GNPTG Normosmic congenital hypogonadotropic hypogonadism 18 GNRH1 Normosmic congenital hypogonadotropic hypogonadism 18 GNRHR Sanfilippo syndrome type D 1 GNS Papillary or follicular thyroid carcinoma 14 GOLGA5 Geroderma osteodysplastica 2 GORAB Progressive myoclonic epilepsy type 6 1 GOSR2 Bernard-Soulier syndrome 3 GP1BA Fetal and neonatal alloimmune thrombocytopenia 6 GP1BA Von Willebrand disease, platelet type 1 GP1BA 22qll.2 deletion syndrome 7 GP1BB Bernard-Soulier syndrome 3 GP1BB Fetal and neonatal alloimmune thrombocytopenia 6 GP1BB Bleeding diathesis due to glycoprotein V I deficiency 1 GP6 Bernard-Soulier syndrome 3 GP9 Biliary atresia 1 GPC1 Simpson-Golabi-Behmel syndrome 2 GPC3 Simpson-Golabi-Behmel syndrome 2 GPC4 Autosomal recessive omodysplasia 1 GPC6 Infantile regressive hypertriglyceridemia and hepatosteatosis 1 GPD1 Brugada syndrome 12 GPD1L

Table IX-21781338.1 Hereditary hyperekplexia 4 GPHN Sulfite oxidase deficiency due to molybdenum cofactor deficiency type C 1 GPHN Hemolytic anemia due t o glucophosphate isomerase deficiency 1 GPI Hyperlipoproteinemia type 1 2 GPIH BP1 Hyperlipoproteinemia type 5 4 GPIHBP1 X-linked recessive ocular albinism 1 GPR143 Congenital stationary night blindness 12 GPR179 Bilateral frontoparietal polymicrogyria 1 GPR56 Usher syndrome type 2 4 GPR98 Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 GPSM2 Chudley-McCullough syndrome 1 GPSM2 Hereditary mixed polyposis syndrome 2 GREM1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GRHL2 Primary hyperoxaluria type 2 1 GRHPR X-linked nonsyndromic intellectual deficit 24 GRIA3 Autosomal recessive nonsyndromic intellectual deficit 15 GRIK2 Autosomal dominant nonsyndromic intellectual deficit 15 GRIN1 Early-onset epileptic encephalopathy and intellectual deficit due to GRIN2A mutation 1 GRIN2A Autosomal dominant nonsyndromic intellectual deficit 15 GRIN2B Fraser syndrome 3 GRIP1 Oguchi disease 2 GRK1 Autosomal recessive congenital cerebellar ataxia due to MGLUR1 deficiency 1 GRM1 Congenital stationary night blindness 12 GRM6 CLN 11 disease 1 GRN Progressive non-fluent aphasia 3 GRN Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 GRXCR1 Familial amyloidosis, Finnish type 1 GSN Hemolytic anemia due to glutathione reductase deficiency 1 GSR Hemolytic anemia due to glutathione reductase deficiency 1 GSR Glutathione synthetase deficiency with 5-oxoprolinuria 1 GSS Glutathione synthetase deficiency without 5-oxoprolinuria 1 GSS Walker-Warburg syndrome 13 GTDC2 (BIDS syndrome 3 GTF2H5 PIBI DS syndrome 3 GTF2H5 Williams syndrome 17 GTF2I Williams syndrome 17 GTF2IRD1 Cone rod dystrophy 22 GUCA1A Progressive cone dystrophy 4 GUCA1A Retinitis pigmentosa 61 GUCA1B

Table IX-21781338.1 Chronic diarrhea due to guanylate cyclase 2C overactivity 1 GUCY2C Intestinal obstruction in the newborn due t o guanylate cyclase 2C deficiency 1 GUCY2C Central areolar choroidal dystrophy 2 GUCY2D Cone rod dystrophy 22 GUCY2D Leber congenital amaurosis 18 GUCY2D Mucopolysaccharidosis type 7 1 GUSB Glycogen storage disease due to glycogenin deficiency 1 GYG1 Glycogen storage disease due t o muscle and heart glycogen synthase deficiency 1 GYS1 Glycogen storage disease due t o hepatic glycogen synthase deficiency 1 GYS2 Beckwith-Wiedemann syndrome due t o llpl5 microdeletion 2 H19 Beckwith-Wiedemann syndrome due t o imprinting defect of llpl5 3 H19 Hemihypertrophy 3 H19 Nephroblastoma 5 H19 Silver-Russell syndrome due t o llpl5 microduplication 2 H19 Silver-Russell syndrome due to llpl5 microduplication 2 H19 Silver-Russell syndrome due to imprinting defect of llpl5 2 H19 Hyperandrogenism due to cortisone reductase deficiency 2 H6PD Neuroblastoma 7 HACE1 Hyperinsulinism due to 3-hydroxylacyl-CoA dehydrogenase deficiency 1 HADH Acute fatty liver of pregnancy 1 HADHA Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency 1 HADHA Mitochondrial trifunctional protein deficiency 2 HADHA Mitochondrial trifunctional protein deficiency 2 HADH B Histidinemia 1 HAL Hemochromatosis type 2 2 HAMP Usher syndrome type 3 3 HARS Perrault syndrome 4 HARS2 Kostmann syndrome 1 HAX1 Alpha thalassemia - intellectual deficit syndrome linked to chromosome 16 2 HBA1 Autosomal dominant methemoglobinemia 3 HBA1 Hb Bart's Hydrops Fetalis 2 HBA1 Hemoglobin H disease 2 HBA1 Alpha thalassemia - intellectual deficit syndrome linked to chromosome 16 2 HBA2 Autosomal dominant methemoglobinemia 3 HBA2 Hb Bart's Hydrops Fetalis 2 HBA2 Hemoglobin H disease 2 HBA2 Autosomal dominant methemoglobinemia 3 HBB Beta-thalassemia intermedia 1 HBB Beta-thalassemia major 1 HBB

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Table IX-21781338.1 Delta-beta thalassemia 3 HBB Dominant beta-thalassemia 1 HBB Heinz body anemia 1 HBB Hemoglobin C - beta-thalassemia 1 HBB Hemoglobin C disease 1 HBB Hemoglobin D disease 1 HBB Hemoglobin E - beta-thalassemia 1 HBB Hemoglobin Lepore - beta-thalassemia 2 HBB Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 HBB Hereditary persistence of fetal hemoglobin - sickle cell disease 4 HBB Sickle cell - beta-thalassemia disease 1 HBB Sickle cell - hemoglobin C disease 1 HBB Sickle cell - hemoglobin D disease 1 HBB Sickle cell - hemoglobin E disease 1 HBB Sickle cell anemia 1 HBB Delta-beta thalassemia 3 HBD Hemoglobin Lepore - beta-thalassemia 2 HBD Delta-beta thalassemia 3 HBG1 Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 HBG1 Hereditary persistence of fetal hemoglobin - sickle cell disease 4 HBG1 Hemoglobinopathy Toms River 1 HBG2 Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 HBG2 Hereditary persistence of fetal hemoglobin - sickle cell disease 4 HBG2 Microphthalmia with linear skin defects syndromes 2 HCCS X-linked nonsyndromic intellectual deficit 24 HCFC1 Brugada syndrome 12 HCN4 Sick sinus syndrome 3 HCN4 Narcolepsy-cataplexy 4 HCRT 2q37 microdeletion syndrome 1 HDAC4 X-linked dominant chondrodysplasia, Chassaing-Lacombe type 1 HDAC6 Cornelia de Lange syndrome 5 HDAC8 Wilson-Turner syndrome 1 HDAC8 Peters anomaly 6 HDAC9 Primary ciliary dyskinesia 2 1 HEATR2 Megalencephalic leukoencephalopathy with subcortical cysts 2 HEPACAM Developmental delay with autism spectrum disorder and gait instability 1 HERC2 Autosomal recessive spondylocostal dysostosis 4 HES7 Combined pituitary hormone deficiencies, genetic forms 5 HESX1 Hypothyroidism due t o deficient transcription factors involved in pituitary development or function 5 HESX1 Kallmann syndrome 19 HESX1

Table IX-21781338.1 Pituitary stalk interruption syndrome 2 HESX1 Septo-optic dysplasia 6 HESX1 Tay-Sachs disease, B variant, adult form 1 HEXA Tay-Sachs disease, B variant, infantile form 1 HEXA Tay-Sachs disease, B variant, juvenile form 1 HEXA Tay-Sachs disease, Bl variant 1 HEXA Sandhoff disease, adult form 1 HEXB Sandhoff disease, adult form 1 HEXB Sandhoff disease, infantile form 1 HEXB Sandhoff disease, juvenile form 1 HEXB Porphyria cutanea tarda 2 HFE Porphyria variegata 2 HFE Hemochromatosis type 2 2 HFE2 Alkaptonuria 1 HGD Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 HGF Sanfilippo syndrome type C 1 HGSNAT Neurodegeneration due to 3-hydroxyisobutyryl-CoA hydrolase deficiency 1 HIBCH Miller-Dieker syndrome 3 HICl Autosomal recessive axonal neuropathy with neuromyotonia 1 HINT1 22qll.2 deletion syndrome 7 HI A Charcot-Marie-Tooth disease type 4G 1 HK1 Nonspherocytic hemolytic anemia due t o hexokinase deficiency 1 HK1 Chorioretinopathy, Birdshot type 1 HLA-A Behcet disease 9 HLA-B Stevens-Johnson syndrome 1 HLA-B Narcolepsy without cataplexy 1 HLA-DQB1 Narcolepsy-cataplexy 4 HLA-DQB1 Graham Little-Piccardi-Lassueur syndrome 1 HLA-DRA Diffuse cutaneous systemic sclerosis 4 HLA-DRB1 Follicular lymphoma 4 HLA-DRB1 Limited cutaneous systemic sclerosis 4 HLA-DRB1 Limited systemic sclerosis 1 HLA-DRBl Narcolepsy-cataplexy 4 HLA-DRB1 Pediatric systemic sclerosis 1 HLA-DRBl Sarcoidosis 2 HLA-DRBl Holocarboxylase synthetase deficiency 1 HLCS Precursor B-cell acute lymphoblastic leukemia 13 HLF Acute intermittent porphyria 1 HMBS 12ql4 microdeletion syndrome 2 HMGA2 Dedifferentiated liposarcoma 3 HMGA2 Well-differentiated liposarcoma 3 H GA2

107

Table IX-21781338.1 3-hydroxy-3-methylglutaric aciduria 1 HMGCL 3-hydroxy 3-methylglutaryl-CoA synthase deficiency 1 HMGCS2 Oculoauricular syndrome, Schorderet type 1 HMX1 Hyperinsulinism due to HNF1A deficiency 1 HNF1A MODY syndrome 12 HNF1A 17ql2 microdeletion syndrome 2 HNF1B Autosomal dominant primary hypomagnesemia with hypocalcuria 2 HNF1B Familial prostate cancer 14 HN F1B Mullerian aplasia 1 HN F1B Renal cysts and diabetes syndrome 1 HNF1B Hyperinsulinism due to HNF4A deficiency 1 HNF4A MODY syndrome 12 H F4A Amyotrophic lateral sclerosis 23 HN RNPA1 Inclusion body myopathy with Paget disease of bone and frontotemporal dementia 2 HNRNPA2B1 Precursor T-cell acute lymphoblastic leukemia 19 HNRNPH1 Primary hyperoxaluria type 3 1 HOGA1 Athabaskan brainstem dysgenesis syndrome 1 HOXA1 Bosley-Salih-Alorainy syndrome 1 HOXA1 Radio-ulnar synostosis - amegakaryocytic thrombocytopenia 1 HOXA11 Radio-ulnar synostosis - amegakaryocytic thrombocytopenia 1 HOXA11 Guttmacher syndrome 1 HOXA13 Hand-foot-genital syndrome 1 HOXA13 Anotia 1 HOXA2 Bilateral microtia - deafness - cleft palate 1 HOXA2 Congenital hereditary facial paralysis with variable hearing loss 1 HOXB1 Familial prostate cancer 14 HOXB13 Pure hair and hail ectodermal dysplasia 2 HOXC13 Congenital vertical talus, bilateral 1 HOXD10 Congenital vertical talus, unilateral 1 HOXD10 Brachydactyly type E 2 HOXD13 Brachydactyly-syndactyly, Zhao type 1 HOXD13 Synpolydactyly type 1 1 HOXD13 VACTERL/VATERassociation 1 HOXD13 Hawkinsinuria 1 HPD Tyrosinemia type 3 1 HPD Cranio-osteoarthropathy 1 HPGD Isolated congenital digital clubbing 1 HPGD Pachydermoperiostosis 2 HPGD Kelley-Seegmiller syndrome 1 HPRT1 Lesch-Nyhan syndrome 1 HPRT1

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Table IX-21781338.1 Hermansky-Pudlak syndrome with pulmonary fibrosis 2 HPS1 Hermansky-Pudlak syndrome without pulmonary fibrosis 3 HPS3 Hermansky-Pudlak syndrome with pulmonary fibrosis 2 HPS4 Hermansky-Pudlak syndrome without pulmonary fibrosis 3 HPS5 Hermansky-Pudlak syndrome without pulmonary fibrosis 3 HPS6 Ochoa syndrome 2 HPSE2 Alopecia universalis 1 HR Atrichia with papular lesions 1 HR Marie Unna hereditary hypotrichosis 2 HR Costello syndrome 2 HRAS Hereditary thrombophilia due t o congenital histidine-rich (poly-L) glycoprotein deficiency 1 HRG Familial isolated hyperparathyroidism 3 HRPT1 Kallmann syndrome 19 HS6ST1 Normosmic congenital hypogonadotropic hypogonadism 18 HS6ST1 Hyperandrogenism due to cortisone reductase deficiency 2 HSD11B1 Apparent mineralocorticoid excess 1 HSD11B2 Intellectual deficit, X-linked - choreoathetosis - abnormal behavior 1 HSD17B10 Short chain 3-hydroxyacyl-CoA dehydrogenase deficiency 1 HSD17B10 46,XY disorder of sex development due to 17-beta-hydroxysteroid dehydrogenase 3 deficiency 1 HSD17B3 Familial prostate cancer 14 HSD17B3 Bifunctional enzyme deficiency 2 HSD17B4 Perrault syndrome 4 HSD17B4 Congenital adrenal hyperplasia due to 3-beta-hydroxysteroid dehydrogenase deficiency 1 HSD3B2 . Familial prostate cancer 14 HSD3B2 Congenital bile acid synthesis defect type 1 1 HSD3B7 Zonular cataract 9 HSF4 Autosomal dominant Charcot-Marie-Tooth disease type 2 F 1 HSPB1 Distal hereditary motor neuropathy type 2 3 HSPB1 Distal hereditary motor neuropathy type 2 3 HSPB3 Autosomal dominant Charcot-Marie-Tooth disease type 2L 1 HSPB8 Distal hereditary motor neuropathy type 2 3 HSPB8 Familial renal cell carcinoma 6 HSPBAP1 Autosomal dominant spastic paraplegia type 13 1 HSPD1 Pelizaeus-Merzbacher-like due to HSPD1 mutation 1 HSPD1 Dyssegmental dysplasia, Silverman-Handmaker type 1 HSPG2 Schwartz-Jampel syndrome 1 HSPG2 CARASIL 1 HTRA1 Young adult-onset Parkinsonism 13 HTRA2 Huntington disease 1 HTT 109

Table IX-21781338.1 Juvenile Huntington disease 1 HTT Intellectual deficit, X-linked, Turner type 1 HUWE1 Hyaluronidase deficiency 1 HYAL1 Primary ciliary dyskinesia 21 HYDIN Hydrolethalus 2 HYLS1 Paternal uniparental disomy of chromosome 6 2 HYMAI Transient neonatal diabetes mellitus 5 HYMAI Chronic mucocutaneous candidiasis 6 ICAM1 Endocrine-cerebro-osteodysplasia syndrome 1 ICK Endocrine-cerebro-osteodysplasia syndrome 1 ICK Common variable immunodeficiency 10 ICOS Enchondromatosis 3 IDH1 Giant cell glioblastoma 10 IDH1 Gliosarcoma 10 IDH1 Maffucci syndrome 2 IDH1 Metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria 1 IDH1 D-2-hydroxyglutaric aciduria 2 IDH2 Enchondromatosis 3 IDH2 Maffucci syndrome 2 IDH2 Retinitis pigmentosa 61 IDH3B Mucopolysaccharidosis type 2A 1 IDS Mucopolysaccharidosis type 2B 1 IDS Hurler syndrome 1 IDUA Hurler-Scheie syndrome 1 IDUA Scheie syndrome 1 IDUA Primary microcephaly-epilepsy-permanent neonatal diabetes syndrome 1 IER3IP1 Osteogenesis imperfecta type 5 2 IFITM5 Idiopathic aplastic anemia 5 IFNG Autosomal dominant mendelian susceptibility to mycobacterial diseases due to partial IFNgammaRl deficiency 1 IFNGR1 Autosomal recessive mendelian susceptibility to mycobacterial diseases due to partial IFNgammaRl deficiency 1 IFNGR1 Mendelian susceptibility to mycobacterial diseases due t o complete IFNgammaRl deficiency 1 IFNGR1 Autosomal dominant mendelian susceptibility to mycobacterial diseases due to partial IFNgammaR2 deficiency 1 IFNGR2 Autosomal recessive mendelian susceptibility'to mycobacterial diseases due to partial IFNgammaR2 deficiency 1 IFNGR2 Mendelian susceptibility to mycobacterial diseases due t o complete IFNgammaR2 deficiency 1 IFNGR2 Spinocerebellar ataxia type 18 1 IFRD1 Cranioectodermal dysplasia 4 IFT122

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Table IX-21781338.1 Jeune syndrome 6 IFT140 Saldino-Mainzer syndrome 1 IFT140 Saldino-Mainzer syndrome 1 IFT140 Cranioectodermal dysplasia 4 IFT43 Jeune syndrome 6 IFT80 Short rib-polydactyly syndrome, Verma-Naumoff type 3 IFT80 Jeune syndrome 6 IFT88 Meckel syndrome 13 IFT88 Agenesis of the corpus callosum - intellectual deficit - coloboma - micrognathia 1 IGBP1 Growth delay due t o insulin-like growth factor 1deficiency 1 IGF1 Growth delay due t o insulin-like growth factor 1resistance 1 IGF1R Beckwith-Wiedemann syndrome due t o imprinting defect of llpl5 3 IGF2 Hemihypertrophy 3 IGF2 Silver-Russell syndrome due to llpl5 microduplication 2 IGF2 Silver-Russell syndrome due to imprinting defect of llplS 2 IGF2 Short stature due t o primary acid-labile subunit deficiency 1 IGFALS Familial retinal arterial macroaneurysm 1 IGFBP7 , Follicular lymphoma 4 IGH@ MALT lymphoma 4 IGH@ Mantle cell lymphoma 3 IGH@ Precursor B-cell acute lymphoblastic leukemia 13 IGH@ B-cell chronic lymphocytic leukemia 7 IGHG1 Recurrent infections associated with rare immunoglobulin isotypes deficiency 2 IGHG2 Autosomal agammaglobulinemia 7 IGHM Spinal muscular atrophy with respiratory distress 2 IGHMBP2 B-cell chronic lymphocytic leukemia 7 IGHV3-21 Hairy cell leukemia variant 1 IGHV4-34 Recurrent infections associated with rare immunoglobulin isotypes deficiency 2 IGKC Autosomal agammaglobulinemia 7 IGLL1 X-linked central congenital hypothyroidism with late-onset testicular enlargement 1 IGSF1 X-linked central congenital hypothyroidism with late-onset testicular enlargement 1 IGSF1 Acrocapitofemoral dysplasia 1 IHH Brachydactyly type Al 2 IHH Familial dysautonomia 1 IKBKAP Anhidrotic ectodermal dysplasia - immunodeficiency - osteopetrosis - lymphedema 1 IKBKG Hypohidrotic ectodermal dysplasia with immunodeficiency 2 IKBKG

Table IX-21781338.1 Incontinentia pigmenti 1 IKBKG X-linked mendelian susceptibility to mycobacterial diseases due to IKBKG deficiency 1 IKBKG Pancytopenia due to IKZF1 mutations 1 IKZF1 Autosomal recessive early-onset inflammatory bowel disease 3 IL10 Behcet disease 9 IL10 Autosomal recessive early-onset inflammatory bowel disease 3 IL10RA Autosomal recessive early-onset inflammatory bowel disease 3 IL10RB Craniosynostosis and dental anomalies 1 IL11RA Primary biliary cirrhosis 8 IL12A Mendelian susceptibility t o mycobacterial diseases due to complete IL12B deficiency 1 IL12B Mendelian susceptibility t o mycobacterial diseases due to complete IL12RB1 deficiency 1 IL12RB1 Primary biliary cirrhosis 8 IL12RB1 Behcet disease 9 IL12RB2 Chronic mucocutaneous candidiasis 6 IL17F Chronic mucocutaneous candidiasis 6 IL17RA Kallmann syndrome 19 IL17RD X-linked nonsyndromic intellectual deficit 24 ILIRAPLI Sterile multifocal osteomyelitis with periostitis and pustulosis 1 IL1RN Cryptosporidiosis - chronic cholangitis - liver disease 1 IL21R Behcet disease 9 IL23R Immunodeficiency due to CD25 deficiency 1 IL2 A Juvenile rheumatoid factor-negative polyarthritis 9 IL2RA Oligoarticular juvenile arthritis 9 IL2RA Juvenile rheumatoid factor-negative polyarthritis 9 IL2RB Oligoarticular juvenile arthritis 9 IL2RB Omenn syndrome 9 IL2RG T-B+ severe combined immunodeficiency due to gamma chain deficiency 1 IL2RG Familial primary localized cutaneous amyloidosis 2 IL31RA Acrodermatitis continua suppurativa of Hallopeau 1 IL36RN Generalized pustular psoriasis 1 IL36RN Pustulosis palmaris et plantaris 1 IL36RN Pustulosis palmaris et plantaris 1 IL36RN Systemic-onset juvenile idiopathic arthritis 2 IL6 Omenn syndrome 9 IL7R T-B+ severe combined immunodeficiency due to IL-7Ralpha deficiency 1 IL7R Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 ILDR1 Catel-Manzke syndrome 1 IMPAD1 Chondrodysplasia with joint dislocations, gPAPP type 1 IM PAD1 Leber congenital amaurosis 18 IMPDH1 112

Table IX-21781338.1 Retinitis pigmentosa 61 IMPDH1 Retinitis pigmentosa 61 IMPG2 Autosomal dominant intermediate Charcot-Marie-Tooth disease type E 1 INF2 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 INF2 Squamous cell carcinoma of head and neck 4 ING1 Squamous cell carcinoma of head and neck 4 ING3 Joubert syndrome 9 INPP5E MORM syndrome 1 INPP5E Opsismodysplasia 1 INPPL1 MODY syndrome 12 INS Permanent neonatal diabetes mel litus 5 INS Hyperinsulinism due t o INSR deficiency 1 INSR Insulin-resistance syndrome type A 1 INSR Leprechaunism 1 INSR Rabson-Mendenhall syndrome 1 INSR Infantile autosomal recessive medullary cystic kidney disease 3 INVS Senior-Loken syndrome 8 INVS Leber congenital amaurosis 18 IQCB1 Senior-Loken syndrome 8 IQCB1 X-linked nonsyndromic intellectual deficit 24 IQSEC2 Immunodeficiency due t o interleukin-1 receptor-associated kinase-4 deficiency 1 IRAK4 Diffuse cutaneous systemic sclerosis 4 IRF5 Limited cutaneous systemic sclerosis 4 IRF5 Primary biliary cirrhosis 8 IRF5 Autosomal dominant popliteal pterygium syndrome 1 IRF6 Oligodontia 10 IRF6 Van Der Woude syndrome 1 IRF6 Mendelian susceptibility t o mycobacterial diseases due t o partial IRF8 deficiency 1 IRF8 Craniofacial dysplasia-osteopenia syndrome 1 IRX5 Hereditary myopathy with lactic acidosis due t o ISCU deficiency 1 ISCU Mendelian susceptibility t o mycobacterial diseases due t o complete ISG15 deficiency 1 ISG15 Autosomal recessive limb-girdle muscular dystrophy due t o ISPD deficiency 1 ISPD Autosomal recessive limb-girdle muscular dystrophy with cerebellar involvement 1 ISPD Walker-Warburg syndrome 13 ISPD Syndromic multisystem autoimmune disease due t o Itch deficiency 1 ITCH Fetal and neonatal alloimmune thrombocytopenia 6 ITGA2

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Table IX-21781338.1 Fetal and neonatal alloimmune thrombocytopenia 6 ITGA2B Glanzmann thrombasthenia 2 ITGA2B Congenital nephrotic syndrome-interstitial lung disease-epidermolysis bullosa syndrome 1 ITGA3 Junctional epidermolysis bullosa - pyloric atresia 2 ITGA6 Congenital muscular dystrophy with integrin deficiency 1 ITGA7 Leukocyte adhesion deficiency type 1 1 ITGB2 Autosomal dominant macrothrombocytopenia 3 ITGB3 Fetal and neonatal alloimmune thrombocytopenia 6 ITGB3 Glanzmann thrombasthenia 2 ITGB3 Epidermolysis bullosa simplex with pyloric atresia 2 ITGB4 Generalized junctional epidermolysis bullosa, non-Herlitz type 5 ITGB4 Junctional epidermolysis bullosa - pyloric atresia 2 ITGB4 Localized junctional epidermolysis bullosa, non-Herlitz type 2 ITGB4 Autosomal recessive lymphoproliferative disease 2 ITK Familial dementia, British type 1 IT 2B Spinocerebellar ataxia type 15/16 1 ITPR1 Spinocerebellar ataxia type 29 1 ITPR1 Spinocerebellar ataxia type 29 1 ITPR1 Isovaleric acidemia 1 IVD Familial thyroid dyshormonogenesis 6 IYD Tetralogy of Fallot 8 JAG1 Budd-Chiari syndrome 2 JAK2 Essential thrombocythemia 4 JAK2 Familial thrombocytosis 3 JAK2 Myelofibrosis with myeloid metaplasia 3 JAK2 Polycythemia vera 3 JAK2 T-B+ severe combined immunodeficiency due to JAK3 deficiency 1 JAK3 Porencephaly-microcephaly-bilateral congenital cataract syndrome 1 JAM3 Huntington disease-like 2 1 JPH3 Childhood absence epilepsy 6 JRK Juvenile myoclonic epilepsy 7 JRK Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 JU P Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 JU P Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 UP Lethal acantholytic epidermolysis bullosa 2 JUP Naxos disease 1 JUP Kallmann syndrome 19 KALI Normosmic congenital hypogonadotropic hypogonadism 18 KALI Familial congenital palsy of trochlear nerve 1 KANK1

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Table IX-21781338.1 Inherited congenital spastic tetraplegia 2 KANK1 17q21.31 microdeletion syndrome 1 KANSL1 Autosomal recessive intermediate Charcot-Marie-Tooth disease type B 1 KARS Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 KARS Blepharophimosis-intellectual deficit syndrome, SBBYS type 1 KAT6B Genitopatellar syndrome 1 KAT6B Genitopatellar syndrome 1 KAT6B Noonan syndrome 10 KAT6B Childhood-onset nemaline myopathy 5 KBTBD13 Episodic ataxia type 1 1 KCNA1 Hereditary continuous muscle fiber activity 1 KCNA1 Isolated autosomal dominant hypomagnesemia, Glaudemans type 1 KCNA1 Familial atrial fibrillation 14 KCNA5 lp36 deletion syndrome 4 KCNAB2 Spinocerebellar ataxia type 13 1 KCNC3 Brugada syndrome 12 KCND3 Spinocerebellar ataxia type 19/22 1 KCND3 Familial atrial fibrillation 14 KCNE1 Jervell and Lange-Nielsen syndrome 2 KCNE1 Romano-Ward syndrome 13 KCN E1 Alport syndrome - intellectual deficit - midface hypoplasia - elliptocytosis 3 KCNE1L Familial atrial fibrillation 14 KCNE2 Romano-Ward syndrome 13 KCNE2 Brugada syndrome 12 KCNE3 Hypokalemic periodic paralysis 3 KCNE3 Familial short T syndrome 4 KCN H2 Romano-Ward syndrome 13 KCN H2 Antenatal Bartter syndrome 2 KCNJ1 Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 KCNJ10 Pendred syndrome 3 KCNJ10 SeSAME syndrome 1 KCNJ10 Autosomal dominant hyperinsulinism due to Kir6.2 deficiency 1 KCNJ11 Autosomal recessive hyperinsulinism due t o Kir6.2 deficiency 1 KCNJ11 DEN D syndrome 1 KCNJ11 Diazoxide-resistant focal hyperinsulinism due t o Kir6.2 deficiency 1 KCNJ11 MODY syndrome 12 KCNJ11 Permanent neonatal diabetes mellitus 5 KCNJ11 Transient neonatal diabetes mellitus 5 KCNJ11 Leber congenital amaurosis 18 KCNJ13 Snowflake vitreoretinal degeneration 1 KCNJ13 Thyrotoxic periodic paralysis 3 KCNJ18

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Table IX-21781338.1 Cardiodysrhythmic potassium-sensitive periodic paralysis 1 KCNJ2 Familial atrial fibrillation 14 KCNJ2 Familial short QT syndrome KCNJ2 Familial hyperaldosteronism type 3 1 KCNJ5 Romano-Ward syndrome 13 KCNJ5 Brugada syndrome 12 KCNJ8 Intellectual deficit, Birk-Barel type 1 KCNK9 Generalized epilepsy - paroxysmal dyskinesia 1 KCNMA1 Familial atrial fibrillation 14 KCNQ1 Familial short QT syndrome 4 KCNQ1 Jervell and Lange-Nielsen syndrome 2 KCNQ1 Romano-Ward syndrome 13 KCNQ1 Beckwith-Wiedemann syndrome due t o llpl5 microdeletion 2 KCNQ10T1 Beckwith-Wiedemann syndrome due t o imprinting defect of llpl5 3 KCNQIOTI Hemihypertrophy 3 KCNQ10T1 Benign familial infantile seizures 4 KCNQ2 Benign familial neonatal seizures 2 KCNQ2 Benign familial neonatal-infantile seizures 2 KCNQ2 Early infantile epileptic encephalopathy 9 KCNQ2 Benign familial infantile seizures 4 KCNQ3 Benign familial neonatal seizures 2 KCNQ3 Juvenile myoclonic epilepsy 7 KCNQ3 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 KCNQ4 Malignant migrating partial seizures of infancy 4 KCNT1 Nocturnal frontal lobe epilepsy 5 KCNT1 Cone dystrophy with supernormal rod response 1 KCNV2 Scalp-ear-nipple syndrome 1 KCTD1 Progressive myoclonic epilepsy type 3 1 KCTD7 Syndromic X-linked intellectual deficit due to JARIDIC mutation 1 KDM5C Kabuki syndrome 2 KDM6A Familial capillary hemangioma 2 KDR Congenital cornea plana 1 KERA Complete hydatidiform mole 2 KHDC3L Essential fructosuria 1 KHK Autosomal dominant spastic paraplegia type 8 1 KIAA0196 Autosomal recessive nonsyndromic intellectual deficit 15 KIAA1033 Goldberg-Shprintzen megacolon syndrome 1 KIAA1279 Monomelic amyotrophy 2 KIAA1377 Pilocytic astrocytoma 2 KIAA1549 Intellectual deficit, X-linked, Cantagrel type 1 KIAA2022 Microcephaly - lymphedema - chorioretinopathy 1 KIF11

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Table IX-21781338.1 Autosomal dominant nonsyndromic intellectual deficit 15 KIF1A Autosomal recessive spastic paraplegia type 30 1 KIF1A Hereditary sensory and autonomic neuropathy type 2 4 KIF1A Autosomal dominant Charcot-Marie-Tooth disease type 2A1 1 IF1B Congenital fibrosis of extraocular muscles 4 KIF21A Spondyloepimetaphyseal dysplasia with multiple dislocations 1 KIF22 Congenital dyserythropoietic anemia type 3 1 KIF23 Autosomal dominant Charcot-Marie-Tooth disease type 2 due t o KIF5A mutation 1 KIF5A Autosomal dominant spastic paraplegia type 10 1 KIF5A Acrocallosal syndrome 2 KIF7 Hydrolethalus 2 KIF7 Joubert syndrome 9 KIF7 Joubert syndrome with ocular defect 5 KIF7 Joubert syndrome with orofaciodigital defect 4 KIF7 Multiple epiphyseal dysplasia, Al-Gazali type 1 KIF7 Autosomal dominant nonsyndromic intellectual deficit 15 KIRREL3 Normosmic congenital hypogonadotropic hypogonadism 18 KISS1 Kallmann syndrome 19 KISS1R Normosmic congenital hypogonadotropic hypogonadism 18 KISS1R Aggressive systemic mastocytosis 1 IT Bullous diffuse cutaneous mastocytosis 1 KIT Cutaneous mastocytoma 1 IT Gastrointestinal stromal tumor 4 KIT Indolent systemic mastocytosis 1 KIT Maculopapular cutaneous mastocytosis 1 KIT Mast cell leukemia 1 KIT Piebaldism 2 KIT . Pseudoxanthomatous diffuse cutaneous mastocytosis 1 KIT Systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disease 1 KIT Telangiectasia macularis eruptiva perstans 1 KIT Familial progressive hyper- and hypopigmentation 1 KITLG Familial progressive hyperpigmentation 1 KITLG Hypercalcemic tumoral calcinosis 3 KL Congenital dyserythropoietic anemia due t o KLF1 mutation 1 KLF1 Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 KLF1 Hereditary persistence of fetal hemoglobin - sickle cell disease 4 KLF1 MODY syndrome 12 KLF11 Pseudohypoaldosteronism type 2D 1 KLHL3 Severe congenital nemaline myopathy 3 KLHL40

Table IX-21781338.1 Retinitis pigmentosa 61 KLHL7 Hypomaturation amelogenesis imperfecta 5 KLK4 Congenital prekallikrein deficiency 1 KLKB1 Hereditary breast cancer 4 KLLN Congenital high-molecular-weight kininogen deficiency 1 KNG1 Cardiofaciocutaneous syndrome 4 KRAS Costello syndrome 2 KRAS Familial pancreatic carcinoma 8 KRAS Juvenile myelomonocytic leukemia 3 KRAS Linear nevus sebaceus syndrome 2 KRAS Linear nevus sebaceus syndrome 2 KRAS Noonan syndrome 10 KRAS Hereditary cerebral cavernous malformation 3 KRIT1 Annular epidermolytic ichthyosis 2 KRT1 Epidermolytic ichthyosis 2 KRT1 Epidermolytic palmoplantar keratoderma 4 KRT1 Ichthyosis hystrix of Curth-Macklin 1 KRT1 Keratosis palmoplantaris striata 3 KRT1 Annular epidermolytic ichthyosis 2 KRT10 Congenital reticular ichthyosiform erythroderma 1 KRT10 Epidermolytic ichthyosis 2 KRT10 Meesmann corneal dystrophy 2 KRT12 White sponge nevus 2 KRT13 Autosomal recessive epidermolysis bullosa simplex 2 KRT14 Dermatopathia pigmentosa reticularis 1 KRT14 Epidermolysis bullosa simplex with mottled pigmentation 2 KRT14 Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT14 Localized epidermolysis bullosa simplex 2 KRT14 Naegeli-Franceschetti-Jadassohn syndrome 1 KRT14 Non-Dowling-Meara generalized epidermolysis bullosa simplex 2 KRT14 Epidermolytic palmoplantar keratoderma 4 KRT16 Pachyonychia congenita 4 KRT16 Pachyonychia congenita 4 KRT17 Sebocystomatosis 1 KRT17 Superficial epidermolytic ichthyosis 1 KRT2 Meesmann corneal dystrophy 2 KRT3 White sponge nevus 2 KRT4 Dowling-Degos disease 2 KRT5 Epidermolysis bullosa simplex with circinate migratory erythema 1 KRT5 Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT5 Localized epidermolysis bullosa simplex 2 KRT5

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Table IX-21781338.1 Non-Dowling-Meara generalized epidermolysis bullosa simplex 2 K T5 Pachyonychia congenita 4 KRT6A Pachyonychia congenita 4 K T6 B Epidermolytic palmoplanar keratoderma 4 KRT6C Woolly hair 4 KRT71 Hypotrichosis simplex of the scalp 2 KRT74 Woolly hair 4 KRT74 Monilethrix 4 KRT81 Monilethrix 4 KRT83 Pure hair and nail ectodermal dysplasia 2 KRT85 Monilethrix 4 KRT86 Epidermolytic palmoplantar keratoderma 4 KRT9 Encephalopathy due to hydroxykynureninuria 1 KYN U Hirschsprung disease 7 L1CAM Hydrocephalus with stenosis of aqueduct of Sylvius 1 L1CAM MASA syndrome 2 L1CAM X-linked complicated corpus callosum dysgenesis 1 L1CAM X-linked complicated spastic paraplegia type 1 1 L1CAM L-2-hydroxyglutaric aciduria 1 L2HGDH Congenital muscular dystrophy type 1A 1 LAMA2 Generalized junctional epidermolysis bullosa, non-Herlitz type 5 LAMA3 Junctional epidermolysis bullosa, Herlitz type 3 LAMA3 LOC syndrome 1 LAMA3 Familial isolated dilated cardiomyopathy 38 LAMA4 Cobblestone lissencephaly without muscular o r ocular involvement 1 LAM B1 LAMB-2-related infantile-onset nephrotic syndrome 1 LAM B2 Pierson syndrome 1 LAM B2 Synaptic congenital myasthenic syndromes 2 LAM B2 Generalized junctional epidermolysis bullosa, non-Herlitz type 5 LAM B3 Junctional epidermolysis bullosa, Herlitz type 3 LAM B3 Generalized junctional epidermolysis bullosa, non-Herlitz type 5 LAMC2 Junctional epidermolysis bullosa inversa 1 LAMC2 Junctional epidermolysis bullosa, Herlitz type 3 LAMC2 Occipital pachygyria and polymicrogyria 1 LAMC3 Glycogen storage disease due to LAM P-2 deficiency 1 LAMP2 Primary immunodeficiency syndrome due to pl4 deficiency 1 LAMTOR2 Cobblestone lissencephaly type B 1 LARG E Congenital muscular dystrophy type I D 1 LARG E Muscle eye brain disease 7 LARGE Walker-Warburg syndrome 13 LARGE Microcephalic primordial dwarfism, Alazami type 1 LARP7

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Table IX-21781338.1 Perrault syndrome 4 LARS2 Williams syndrome 17 LAT2 Greenberg dysplasia 1 LBR Reynolds syndrome 1 LBR Leber congenital amaurosis 18 LCA5 Familial LCAT deficiency 1 LCAT Fish-eye disease 1 LCAT Severe combined immunodeficiency due t o LCK deficiency 1 LCK Congenital lactase deficiency 1 LCT Congenital lactase deficiency 1 LCT Arrhythmogenic right ventricular dysplasia 1 LDB3 Familial isolated dilated cardiomyopathy 38 LDB3 Left ventricular noncompaction 11 LDB3 Tibial muscular dystrophy 2 LDB3 ZASP-related myofibrillar myopathy 1 LDB3 Glycogen storage disease due t o lactate dehydrogenase M-subunit deficiency 1 LDHA Glycogen storage disease due t o lactate dehydrogenase H-subunit deficiency 1 LDH B Situs ambiguus 6 LEFTY2 12ql4 microdeletion syndrome 2 LEMD3 Buschke-Ollendorff syndrome 1 LEMD3 Isolated osteopoikilosis 1 LEMD3 Melorheostosis with osteopoikilosis 1 LEMD3 Obesity due t o congenital leptin deficiency 1 LEP Obesity due t o leptin receptor gene deficiency 1 LEPR Osteogenesis imperfecta type 2 5 LEPRE1 Osteogenesis imperfecta type 3 9 LEPRE1 Rare isolated myopia 1 LEPREL1 Wolf-Hirschhorn syndrome 3 LETM 1 Autosomal recessive spondylocostal dysostosis 4 LFNG Autosomal dominant epilepsy with auditory features 2 LGI1 Leydig cell hypoplasia due t o LHB deficiency 1 LHB Leydig cell hypoplasia due t o complete LH resistance 1 LHCGR Testotoxicosis 1 LHCGR Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 LHFPL5 17ql2 microdeletion syndrome 2 LHX1 Classic Mayer-Rokitansky-Kuster-Hauser syndrome 1 LHX1 MURCS association 1 LHX1 Hypothyroidism due t o deficient transcription factors involved in pituitary development or function 5 LHX3 Nonacquired combined pituitary hormone deficiency with spine 1 LHX3 0

Table IX-21781338.1 abnormalities Hypothyroidism due t o deficient transcription factors involved in pituitary development or function 5 LHX4 Pituitary stalk interruption syndrome 2 LHX4 Short stature - pituitary and cerebellar defects - small sella turcica 1 LHX4 Stiive-Wiedemann syndrome 1 LIFR LIG4 syndrome 1 LIG4 Omenn syndrome 9 LIG4 Pulverulent cataract 7 LIM2 Williams syndrome 17 LIMK1 Neuroblastoma 7 LIN28B Autosomal recessive nonsyndromic intellectual deficit 15 LINS

Cholesteryl ester storage disease 1 LIPA Wolman disease 1 LI PA

Hyperlipidemia due t o hepatic triglyceride lipase deficiency 1 LIPC Hypotrichosis simplex 6 LIPH Woolly hair 4 LIPH Hyperlipoproteinemia type 4 2 LIPI Lamellar ichthyosis 6 LIPN Charcot-Marie-Tooth disease type 1C 1 LITAF Combined deficiency of factor V and factor VIII 2 LMAN 1 Adactyly of foot, bilateral 1 LMBR1 Adactyly of foot, unilateral 1 LMBR1 Hypoplastic tibiae - postaxial Polydactyly 2 LMBR1 Polydactyly of a triphalangeal thumb, bilateral 2 LMBR1 Polydactyly of a triphalangeal thumb, unilateral 2 LMBR1 Radial hemimelia, bilateral 2 LMBR1 Radial hemimelia, unilateral 2 LMBR1 Syndactyly type 4 2 LMBR1 Triphalangeal thumb - polysyndactyly syndrome 2 LMBR1 Methylmalonic acidemia with homocystinuria, type cblF 2 LMBRD1 Hyperlipoproteinemia type 1 2 LMF1 Atypical Werner syndrome 1 LMNA Autosomal codominant severe lipodystrophic laminopathy 1 LMNA Autosomal dominant Emery-Dreifuss muscular dystrophy 4 LMNA Autosomal dominant limb-girdle muscular dystrophy type I B 1 LMNA Autosomal recessive Emery-Dreifuss muscular dystrophy 1 LMNA Charcot-Marie-Tooth disease type 2B1 1 LMNA Congenital muscular dystrophy due to LM NA mutation 1 LMNA Dilated cardiomyopathy - hypergonadotropic hypogonadism 1 LMNA Familial dilated cardiomyopathy with conduction defect due t o LMNA 1 LMNA

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Table IX-21781338.1 mutation Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 LMNA Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 LMNA Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 LMNA Familial partial lipodystrophy, Dunnigan type 1 LMNA Familial partial lipodystrophy, Kobberling type 1 LMNA Heart-hand syndrome, Slovenian type 1 LMNA Hutchinson-Gilford progeria syndrome 2 LMNA Laminopathy type Decaudain-Vigouroux 1 LMNA Left ventricular noncompaction 11 LMNA Lethal restrictive dermopathy 2 LMNA Mandibuloacral dysplasia with type A lipodystrophy 1 LMNA Progeria-associated arthropathy 1 LMNA Adult-onset autosomal dominant leukodystrophy 1 LMN B1 Partial acquired lipodystrophy 1 LMN B2 Nail-patella syndrome 1 LMX1B Keratoderma hereditarium mutilans with ichthyosis 1 LOR Progressive symmetric erythrokeratodermia 1 LOR Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 LOXHD1 Hypotrichosis simplex . 6 LPAR6 Woolly hair 4 LPAR6 Genetic recurrent myoglobinuria 3 LPIN 1 Majeed syndrome 1 LPIN2 Familial lipoprotein lipase deficiency 1 LPL Hyperlipoproteinemia type 5 4 LPL Leber congenital amaurosis 18 LRAT Retinitis pigmentosa 6 1 LRAT Common variable immunodeficiency 10 LRBA Ochoa syndrome 2 LRIG2 Congenital stationary night blindness 12 LRIT3 Donnai-Barrow syndrome 1 LRP2 Cenani-Lenz syndrome 1 LRP4 Sclerosteosis, 2 LRP4 Autosomal dominant osteopetrosis type 1 1 LRP5 Autosomal dominant osteosclerosis, Worth type 1 LRP5 Familial exudative vitreoretinopathy 5 LRP5 Hyperostosis corticalis generalisata 2 LRP5 Idiopathic juvenile osteoporosis 4 LRP5 Osteoporosis - pseudoglioma 1 LRP5 Osteosclerosis - developmental delay - craniosynostosis 1 LRP5

122

Table IX-21781338.1 Retinopathy of prematurity 3 LRP5 Coronary artery disease - hyperlipidemia - hypertension - diabetes - osteoporosis 1 LRP6 Congenital lactic acidosis, Saguenay-Lac-St. Jean type 1 LRPPRC Primary ciliary dyskinesia 21 LRRC50 Primary ciliary dyskinesia 21 LRRC6 Autosomal agammaglobulinemia 7 LRRC8A Hereditary nonpolyposis colon cancer 11 LRRFIP2 Young adult-onset Parkinsonism 13 LRRK2 Autosomal dominant Charcot-Marie-Tooth disease type 2P 1 LRSAM1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 LRTOMT Congenital glaucoma 3 LTBP2 Glaucoma secondary t o spherophakia/ectopia lentis and megalocornea 1 LTBP2 Weill-Marchesani syndrome 4 LTBP2 Oligodontia 10 LTBP3 Cutis laxa with severe pulmonary, gastrointestinal and urinary anomalies 1 LTBP4 Duchenne muscular dystrophy 2 LTBP4 Hypotonia - failure to thrive - microcephaly 1 LTC4S Attenuated Chediak-Higashi syndrome 1 LYST Chediak-Higashi syndrome 1 LYST Familial renal amyloidosis due t o lysozyme variant 1 LYZ Bardet-Biedl syndrome 17 LZTFL1 Cataract-microcornea syndrome 8 MAF Cerulean cataract 4 MAF Pulverulent cataract 7 MAF Multicentric carpo-tarsal osteolysis with or without nephropathy 1 MAFB Prader-Willi syndrome 6 MAGEL2 West syndrome 4 MAGI2 X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasia 1 MAGT1 X-linked nonsyndromic intellectual deficit 24 MAGT1 Retinitis pigmentosa 61 MAK MALT lymphoma 4 MALT1 Familial hypospadias 2 MAMLD1 X-linked centronuclear myopathy 2 MAMLD1 Autosomal recessive nonsyndromic intellectual deficit 15 MAN1B1 Alpha-mannosidosis, adult form 1 MAN2B1 Alpha-mannosidosis, infantile form 1 MAN2B1 Beta-mannosidosis 1 MANBA Familial pancreatic carcinoma 8 MANF Monoamine oxidase-A deficiency 1 MAOA

123

Table IX- 21781338.1 Cardiofaciocutaneous syndrome 4 MAP2K1 Cardiofaciocutaneous syndrome 4 MAP2K2 46,XY complete gonadal dysgenesis 8 MAP3K1 46,XY partial gonadal dysgenesis 7 MAP3K1 Distal 22qll.2 microdeletion syndrome 3 MAPK1 Lennox-Gastaut syndrome 2 MAPK10 Behavioral variant o f frontotemporal dementia 3 MAPT Classical progressive supranuclear palsy 1 MAPT Progressive non-fluent aphasia 3 MAPT Progressive supranuclear palsy - corticobasal syndrome 1 MAPT Progressive supranuclear palsy - parkinsonism 1 MAPT Progressive supranuclear palsy - progressive non fluent aphasia 1 MAPT Progressive supranuclear palsy - pure akinesia with gait freezing 1 MAPT Semantic dementia 2 MAPT Autosomal recessive spastic ataxia with leukoencephalopathy 1 MARS2 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MARVELD2 Craniofacial-ulnar-renal syndrome 2 MASP1 Immunodeficiency due t o MASP-2 deficiency 1 MASP2 Autosomal thrombocytopenia with normal platelets 3 MASTL Brain demyelination due t o methionine adenosyltransferase deficiency 1 MAT1A Multiple epiphyseal dysplasia type 5 1 MATN3 Spondyloepimetaphyseal dysplasia, matrilin-3 type 1 MATN3 Distal myopathy with vocal cord weakness 1 MATR3 Hereditary pheochromocytoma-paraganglioma 7 MAX 2q23.1 microdeletion syndrome 1 MBD5 Autosomal dominant nonsyndromic intellectual deficit 15 MBD5 BRESEK syndrome 1 MBTPS2 Ichthyosis follicularis - alopecia - photophobia 1 MBTPS2 Keratosis follicularis spinulosa decalvans 2 MBTPS2 Mutilating palmoplantar keratoderma with periorificial keratotic plaques 2 MBTPS2 Familial melanoma 6 MC1R Large congenital melanocytic nevus 1 MC1R Oculocutaneous albinism type 2 2 MC1R Familial glucocorticoid deficiency 4 MC2R Obesity due M C3 deficiency 1 MC3R Obesity due t o melanocortin-4 receptor deficiency 1 MC4R Isolated 3-methylcrotonyl-CoA carboxylase deficiency 2 MCCC1 Isolated 3-methylcrotonyl-CoA carboxylase deficiency 2 MCCC2 Methylmalonic acidemia due t o methylmalonyl-CoA epimerase deficiency 1 MCEE Combined deficiency of factor V and factor VII I 2 MCFD2 Immunodeficiency with natural-killer cell deficiency 1 MCM4

124

Table IX-21781338.1 Mucolipidosis type 4 1 MCOLN1 Autosomal recessive primary microcephaly 11 MCPH1 Premature chromosome condensation with microcephaly and intellectual deficit 1 MCPH1 Dedifferentiated liposarcoma 3 MDM2 Well-differentiated liposarcoma 3 MDM2 Myelodysplastic syndromes 2 MECOM Atypical Rett syndrome 4 MECP2 Intellectual deficit, X-linked - psychosis - macroorchidism 1 MECP2 Rett syndrome 1 MECP2 Severe neonatal-onset encephalopathy with microcephaly 1 MECP2 ^Trisomy Xq28 1 MECP2 X-linked nonsyndromic intellectual deficit 24 MECP2 Blepharophimosis-intellectual deficit syndrome, MKB type 1 MED12 FG syndrome type 1 1 MED12 X-linked intellectual deficit with marfanoid habitus 3 MED12 X-linked nonsyndromic intellectual deficit 24 MED12 Autosomal recessive nonsyndromic intellectual deficit 15 MED23 Charcot-Marie-Tooth disease type 2B2 1 MED25 5ql4.3 microdeletion syndrome 1 MEF2C Behcet disease 9 MEFV Familial mediterranean fever 1 MEFV Intermittent hydrarthrosis 2 MEFV Maternal uniparental disomy of chromosome 14 3 MEG3 · Paternal uniparental disomy of chromosome 14 3 MEG3 Spinal muscular atrophy with respiratory distress 2 MEGF10 Carpenter syndrome 2 MEGF8 Familial isolated hyperparathyroidism 3 MEN 1 Familial parathyroid/adenoma 2 MEN 1 Multiple endocrine neoplasia type 1 5 MEN 1 Zollinger-Ellison syndrome 1 MEN 1 Zollinger-Ellison syndrome 1 MEN 1 Isolated Klippel-Feil syndrome 3 MEOX1 Retinitis pigmentosa 61 M E TK Autosomal recessive spondylocostal dysostosis 4 MESP2 Familial papillary renal cell carcinoma 1 MET Hepatocellular carcinoma, childhood-onset 2 MET Autosomal dominant Charcot-Marie-Tooth disease type 2A2 1 MFN2 Autosomal dominant optic atrophy plus syndrome 2 MFN2 Hereditary motor and sensory neuropathy type 5 1 MFN2 Hereditary motor and sensory neuropathy type 6 1 MFN2

Table IX-21781338.1 Severe early-onset axonal neuropathy due to M FN2 deficiency 1 MFN2 Microphthalmia - retinitis pigmentosa - foveoschisis - optic disc drusen 1 M F P Nanophthalmia 1 MFRP CLN7 disease 1 MFSD8 MGAT2-CDG syndrome 1 MGAT2 Progressive external ophthalmoplegia - myopathy - emaciation 1 MGME1 Giant cell glioblastoma 10 MGMT Gliosarcoma 10 MGMT Keutel syndrome 1 MGP Left ventricular noncompaction 11 MIB1 X-linked Opitz G/BBB syndrome 1 MIDI Systemic-onset juvenile idiopathic arthritis 2 MIF Cataract with Y-shaped suture opacities 3 MIP Cerulean cataract 4 MIP Nuclear cataract 9 MIP Total congenital cataract 5 MIP Zonular cataract 9 MIP Laurin-Sandrow syndrome 1 MIPOL1 Feingold syndrome 3 MIR17HG EDICT syndrome 1 MIR184 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MIR96 Clear cell renal carcinoma 1 MITF M ITF-related melanoma and renal cell carcinoma predisposition syndrome 1 MITF Ocular albinism with congenital sensorineural deafness 2 MITF Papillary renal cell carcinoma 1 MITF Tietz syndrome 1 MITF Waardenburg syndrome type 2 4 MITF Bardet-Biedl syndrome 17 MKKS Laurence-Moon syndrome 1 MKKS McKusick-Kaufman syndrome 1 MKKS Bardet-Biedl syndrome 17 MKS1 Meckel syndrome 13 MKS1 Megalencephalic leukoencephalopathy with subcortical cysts 2 MLC1 Constitutional mismatch repair deficiency syndrome 4 MLH1 Hereditary nonpolyposis colon cancer 11 MLH1 Muir-Torre syndrome 3 MLH1 Nonpolyposis Turcot syndrome 4 MLH1 Hereditary nonpolyposis colon cancer 11 MLH3 Acute biphenotypic leukemia 2 MLL Acute myeloid leukemia with llq23 abnormalities 1 MLL Acute undifferentiated leukemia 1 MLL

126

Table IX-21781338.1 Bilineal acute leukemia 1 MLL Precursor B-cell acute lymphoblastic leukemia 13 MLL Wiedemann-Steiner syndrome 1 MLL Kabuki syndrome 2 MLL2 Precursor T-cell acute lymphoblastic leukemia 19 MLLT10 Griscelli disease type 3 2 MLPH Williams syndrome 17 MLXIPL Malonic aciduria 1 MLYCD Vitamin B12-responsive methylmalonic acidemia type cbIA 1 MMAA Methylmalonic acidemia with homocystinuria, type cbIC 1 MMACHC Methylcobalamin deficiency type cblDvl 1 MMADHC Vitamin B12-responsive methylmalonic acidemia, type cblDv2 1 MMADHC Primary biliary cirrhosis 8 M M EL1 Severe generalized recessive dystrophic epidermolysis bullosa 2 MMP1 Metaphyseal anadysplasia 2 MMP13 Spondyloepimetaphyseal dysplasia, Missouri type 1 MMP13 Torg-Winchester syndrome 2 MMP14 Nodulosis-arthropathy-osteolysis syndrome 1 MMP2 Torg-Winchester syndrome 2 MMP2 Hypomaturation amelogenesis imperfecta 5 MMP20 Metaphyseal anadysplasia 2 MMP9 Familial multiple meningioma 5 M N1 Currarino triad 1 MNX1 Xanthinuria type II 1 MOCOS Sulfite oxidase deficiency due to molybdenum cofactor deficiency type A 1 MOCS1 Sulfite oxidase deficiency due to molybdenum cofactor deficiency type B 1 MOCS2 Sulfite oxidase deficiency due to molybdenum cofactor deficiency 1 MOCS3 Narcolepsy-cataplexy 4 MOG GCS1-CDG syndrome 1 MOGS MPDU1-CDG syndrome 1 MPDU1 Congenital communicating hydrocephalus 1 MPDZ MPI-CDG syndrome 1 MPI Congenital amegakaryocytic thrombocytopenia 1 MPL Essential thrombocythemia 4 MPL Familial throm bocytosis 3 MPL Myelofibrosis with myeloid metaplasia 3 MPL Polycythemia vera 3 MPL BIDS syndrome 1 MPLKIP Myeloperoxidase deficiency 1 MPO Encephalopathy due to beta-mercaptolactate-cysteine disulfiduria 1 MPST Navajo neurohepatopathy 1 MPV17

127

Table IX-21781338.1 Autosomal dominant Charcot-Marie-Tooth disease type 2 1 1 MPZ Autosomal dominant Charcot-Marie-Tooth disease type 2J 1 MPZ Autosomal dominant intermediate Charcot-Marie-Tooth disease type D 1 MPZ Autosomal dominant intermediate Charcot-Marie-Tooth disease with neuropathic pain 1 MPZ Charcot-Marie-Tooth disease type I B 1 MPZ Dejerine-Sottas syndrome 4 MPZ Roussy-Levy syndrome 2 MPZ Familial glucocorticoid deficiency 4 MRAP Ataxia-telangiectasia-like disorder 1 MRE11A Hereditary breast and ovarian cancer syndrome 13 MRE11A Combined oxidative phosphorylation defect type 9 1 MRPL3 Infantile hypertrophic cardiomyopathy due to MRPL44 deficiency 1 MRPL44 Combined oxidative phosphorylation defect type 2 1 MRPS16 Hypotonia with lactic acidemia and hyperammonemia 1 MRPS22 Common variable immunodeficiency 10 MS4A1 Constitutional mismatch repair deficiency syndrome 4 MSH2 Hereditary nonpolyposis colon cancer 11 MSH2 Muir-Torre syndrome 3 MSH2 Nonpolyposis Turcot syndrome 4 MSH2 Constitutional mismatch repair deficiency syndrome 4 MSH6 Hereditary nonpolyposis colon_cancer 11 MSH6 Muir-Torre syndrome 3 MSH6 Nonpolyposis Turcot syndrome 4 MSH6 Familial prostate cancer 14 M5MB Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MSRB3 Myostatin-related muscle hypertrophy 1 MSTN Hypodontia - dysplasia of nails 1 MSX1 Oligodontia 10 MSX1 Craniosynostosis, Boston type 1 M5X2 Parietal foramina 2 MSX2 Parietal foramina with cleidocranial dysplasia 1 MSX2 Diaphyseal medullary stenosis - bone malignancy 1 MTAP Familial infantile bilateral striatal necrosis 2 MT-ATP6 Leber hereditary optic neuropathy 11 MT-ATP6 Maternally-inherited Leigh syndrome 14 MT-ATP6 Maternally-inherited spastic paraplegia 1 MT-ATP6 NARP syndrome 1 MT-ATP6 Kearns-Sayre syndrome 3 MT-ATP8 Genetic recurrent myoglobinuria 3 MT-COl Isolated cytochrome C oxidase deficiency 9 MT-COl

128

Table IX-21781338.1 Leber hereditary optic neuropathy 11 MT-COl Maternally-inherited Leigh syndrome 14 MT-COl MELAS syndrome 13 MT-COl Mitochondrial nonsyndromic sensorineural deafness 6 MT-COl Mitochondrial nonsyndromic sensorineural deafness with susceptibility t o aminoglycoside exposure 4 MT-COl Isolated cytochrome C oxidase deficiency 9 MT-C02 Maternally-inherited Leigh syndrome 14 MT-C02 MELAS syndrome 13 MT-C02 Genetic recurrent myoglobinuria 3 MT-C03 Isolated cytochrome C oxidase deficiency 9 MT-C03 Leber hereditary optic neuropathy 11 MT-C03 Maternally-inherited Leigh syndrome 14 MT-C03 M ELAS syndrome 13 MT-C03 Histiocytoid cardiomyopathy 1 MT-CYB Isolated CoQ-cytochrome C reductase deficiency 6 MT-CYB Leber hereditary optic neuropathy 11 MT-CYB Combined oxidative phosphorylation defect type 15 1 MTFMT Isolated NADH-CoQ reductase deficiency 25 MTFMT Cervical spina bifida aperta 6 MTH FD1 Cervical spina bifida cystica 6 MTH FD1 Cervicothoracic spina bifida aperta 6 MTHFD1 Cervicothoracic spina bifida cystica 6 MTHFD1 Lumbosacral spina bifida aperta 6 MTHFD1 Lumbosacral spina bifida cystica 6 MTH FD1 Thoracolumbosacral spina bifida aperta 6 MTH FD1 Thoracolumbosacral spina bifida cystica 6 MTHFD1 Total spina bifida aperta 6 MTHFD1 Total spina bifida cystica 6 MTHFD1 Upper thoracic spina bifida aperta 6 MTHFD1 . Upper thoracic spina bifida cystica 6 MTH FD1 Cervical spina bifida aperta 6 MTH Cervical spina bifida cystica 6 MTH FR Cervicothoracic spina bifida aperta 6 MTH F Cervicothoracic spina bifida cystica 6 MTHFR Homocystinuria due t o methylenetetrahydrofolate reductase deficiency 1 MTH FR Isolated anencephaly/exencephaly 2 MTHFR Lumbosacral spina bifida aperta 6 MTHFR Lumbosacral spina bifida cystica 6 MTHFR Methotrexate poisoning 1 MTHFR Thoracolumbosacral spina bifida aperta 6 MTH FR

1 9

Table IX-21781338.1 Thoracolumbosacral spina bifida cystica 6 MTHFR Total spina bifida aperta 6 MTHFR Total spina bifida cystica 6 MTHFR Upper thoracic spina bifida aperta 6 MTHFR Upper thoracic spina bifida cystica 6 MTHFR X-linked centronuclear myopathy 2 MTM1 Autosomal dominant centronuclear myopathy 4 MTMR14 Leber hereditary optic neuropathy 11 MT-ND1 Maternally-inherited Leigh syndrome 14 MT-ND1 M ELAS syndrome 13 MT-ND1 Isolated NADH-CoQ reductase deficiency 25 MT-N D2 Leber hereditary optic neuropathy 11 MT-ND2 Maternally-inherited Leigh syndrome 14 MT-ND2 Leber hereditary optic neuropathy . 11 MT-ND3 Leber 'plus' disease 3 MT-ND3 Maternally-inherited Leigh syndrome 14 MT- D3 Sporadic Leigh syndrome 3 MT-ND3 Leber hereditary optic neuropathy 11 MT-ND4 Leber 'plus' disease 3 MT-ND4 Maternally-inherited Leigh syndrome 14 MT- D4 M ELAS syndrome 13 MT-ND4 Mitochondrial nonsyndromic sensorineural deafness with susceptibility t o aminoglycoside exposure 4 MT-ND4 Leber hereditary optic neuropathy 11 MT-ND4L Leber hereditary optic neuropathy 11 MT-ND5 Maternally-inherited Leigh syndrome 14 MT-ND5 M ELAS syndrome 13 MT-ND5 M E RF syndrome 10 MT-ND5 Sporadic Leigh syndrome 3 MT-ND5 Leber hereditary optic neuropathy 11 MT-ND6 Leber 'plus' disease 3 MT-ND6 Maternally-inherited Leigh syndrome 14 MT-N D6 M ELAS syndrome 13 MT-ND6 Sporadic Leigh syndrome 3 MT-ND6 Mitochondrial hypertrophic cardiomyopathy with lactic acidosis due t o MTOl deficiency 1 MTOl Autosomal recessive spastic ataxia - optic atrophy - dysarthria 1 MTPAP Methylcobalamin deficiency type cbIG 1 MTR MERRF syndrome 10 MT-RNR1 Mitochondrial nonsyndromic sensorineural deafness 6 MT-RNR1 Mitochondrial nonsyndromic sensorineural deafness with susceptibility to aminoglycoside exposure 4 MT-RN R1 130 '

Table IX-21781338.1 Methylcpbalamin deficiency type cblE 1 MTRR Maternally inherited diabetes and deafness 3 MT-TE Mitochondrial myopathy with reversible cytochrome C oxidase deficiency 2 MT-TE Myopathy and diabetes mellitus 1 MT-TE M ELAS syndrome 13 MT-TF M E RF syndrome 10 MT-TF Maternally-inherited mitochondrial hypertrophic cardiomyopathy 2 MT-TG MELAS syndrome 13 MT-TH MERRF syndrome 10 MT-TH Mitochondrial nonsyndromic sensorineural deafness 6 MT-TH Maternally-inherited mitochondrial hypertrophic cardiomyopathy 2 MT-TI Maternally inherited diabetes and deafness 3 MT-TK Maternally-inherited cardiomyopathy and hearing loss 1 MT-TK Maternally-inherited Leigh syndrome 14 MT-TK MERRF syndrome 10 MT-TK Hypertrophic cardiomyopathy and renal tubular disease due t o mitochondrial DNA mutation 1 MT-TL1 Kearns-Sayre syndrome 3 MT-TL1 Maternally inherited diabetes and deafness 3 MT-TL1 Maternally-inherited Leigh syndrome 14 MT-TL1 Maternally-inherited progressive external ophthalmoplegia 1 MT-TL1 MELAS syndrome 13 MT-TL1 MERRF syndrome 10 MT-TL1 Endomyocardial fibroelastosis 1 MT-TL2 Abetalipoproteinemia 2 MTTP M ERRF syndrome 10 MT-TP M ELAS syndrome 13 MT-TQ MERRF syndrome 10 MT-TQ MELAS syndrome 13 MT-TS1 MERRF syndrome 10 MT-TS1 Mitochondrial nonsyndromic sensorineural deafness 6 MT-TS1 Mitochondrial nonsyndromic sensorineural deafness with susceptibility to aminoglycoside exposure 4 MT-TS1 Palmoplantar keratoderma-deafness syndrome 2 MT-TS1 M ELAS syndrome 13 MT-TS2 MERRF syndrome 10 MT-TS2 Usher syndrome type 3 3 MT-TS2 Lethal infantile mitochondrial myopathy 1 MT-TT Maternally-inherited Leigh syndrome 14 MT-TV Maternally-inherited Leigh syndrome 14 MT-TW Autosomal dominant medullary cystic kidney disease with or without hyperuricemia 2 MUC1

Table IX-21781338.1 Diffuse panbronchiolitis 2 MUC5B Idiopathic pulmonary fibrosis 11 MUC5B Postsynaptic congenital myasthenic syndromes 9 MUSK Vitamin B12-unresponsive methylmalonic acidemia type mut- 1 MUT Vitamin B12-unresponsive methylmalonic acidemia type mutO 1 MUT Familial gastric cancer 2 MUTYH MUTYH-related attenuated familial adenomatous polyposis 1 MUTYH Disseminated superficial actinic porokeratosis 1 MVK Hyperimmunoglobulinemia D with periodic fever 1 MVK Mevalonic aciduria 1 MVK Familial prostate cancer 14 MXI1 Acute basophilic leukemia 2 MYB Precursor T-cell acute lymphoblastic leukemia 19 MYB " Digitotalar dysmorphism 5 MYBPC1 Lethal congenital contracture syndrome type 3 2 MYBPC1 Familial isolated dilated cardiomyopathy 38 MYBPC3 Left ventricular noncompaction 11 MYBPC3 Burkitt lymphoma 1 MYC Precursor T-cell acute lymphoblastic leukemia 19 MYC Feingold syndrome 3 MYCN Neuroblastoma 7 MYCN Pyogenic bacterial infections due to MyD88 deficiency 1 MYD88 Waldenstrom macroglobulinemia 1 MYD88 Autosomal dominant centronuclear myopathy 4 MYF6 Acute myeloid leukemia with abnormal bone marrow eosinophils inv(16)(pl3q22) or t (16;16)(pl3;q22) 2 MYH11 Familial aortic dissection 1 MYH11 Familial thoracic aortic aneurysm and aortic dissection 8 MYH11 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MYH14 Hereditary inclusion body myopathy - joint contractures - ophthalmoplegia 1 MYH2 Digitotalar dysmorphism 5 MYH3 Freeman-Sheldon syndrome 1 MYH3 Sheldon-Hall syndrome 4 MYH3 Atrial septal defect, ostium secundum type 8 MYH6 Familial isolated dilated cardiomyopathy 38 MYH6 Sick sinus syndrome 3 MYH6 Classic multiminicore myopathy 2 MYH7 Familial isolated dilated cardiomyopathy 38 MYH7 Hyaline body myopathy 1 MYH7 Laing distal myopathy 1 MYH7 Left ventricular noncompaction 11 MYH7

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Table IX-21781338.1 Scapuloperoneal amyotrophy 4 MYH7 Carney complex-trismus-pseudocamptodactyly syndrome 1 MYH8 Trismus - pseudocamptodactyly 2 MYH8 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MYH9 Congenital fiber-type disproportion myopathy 5 MYL2 Familial thoracic aortic aneurysm and aortic dissection 8 MYLK Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY015A Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MYOIA Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 MYOIE Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 MY03A Griscelli disease type 1 1 MY05A Griscelli disease type 3 2 MY05A Neuroectodermal melanolysosomal disease 1 MY05A Microvillous inclusion disease 1 MY05B Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MY06 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY06 Progressive sensorineural hearing loss - hypertrophic cardiomyopathy 1 MY06 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MY07A Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY07A Usher syndrome type 1 9 MY07A Congenital glaucoma 3 MYOC Juvenile glaucoma 2 MYOC Autosomal dominant limb-girdle muscular dystrophy type 1A 1 MYOT Myotilin-related myofibrillar myopathy without spheroid body 1 MYOT Spheroid body myopathy 1 MYOT Familial isolated dilated cardiomyopathy 38 MYPN Familial isolated restrictive cardiomyopathy 3 MYPN Premature ageing appearance-developmental delay-cardiac arrhythmia syndrome 1 NAA10 Solitary fibrous tumor 2 NAB2 . Acute promyelocytic leukemia 4 NABP1 Alpha-N-acetylgalactosaminidase deficiency type 1 1 NAGA Sanfilippo syndrome type B 1 NAGLU Hyperammonemia due to N-acetylglutamate synthetase deficiency 1 NAGS Proximal spinal muscular atrophy type 1 3 NAIP Proximal spinal muscular atrophy type 2 3 NAIP Proximal spinal muscular atrophy type 3 3 NAIP Male infertility due to NANOS1 mutation 1 NANOS1 Gray platelet syndrome 1 NBEAL2 Familial prostate cancer 14 NBN

Table IX-21781338.1 Hereditary breast and ovarian cancer syndrome 13 NBN Nijmegen breakage syndrome 1 NBN Papillary or follicular thyroid carcinoma 14 NCOA4 Hydranencephaly 1 NDE1 Microlissencephaly 1 NDE1 Prader-Willi syndrome 6 NDN Coats disease 1 NDP Familial exudative vitreoretinopathy 5 NOP Norrie disease 2 NDP Persistent hyperplastic primary vitreous 3 NDP Retinopathy of prematurity 3 NDP Charcot-Marie-Tooth disease type 4D 1 ND G1 Isolated NADH-CoQ reductase deficiency 25 NDUFA1 Leigh syndrome with leukodystrophy 13 NDUFA10 Isolated NADH-CoQ reductase deficiency 25 NDUFA11 Isolated NADH-CoQ reductase deficiency 25 NDUFA2 Leigh syndrome with leukodystrophy 13 NDUFA2 Isolated NADH-CoQ reductase deficiency 25 NDUFA9 Fatal infantile hypertrophic cardiomyopathy due to mitochondrial complex 1deficiency 1 NDUFAF1 Isolated NADH-CoQ reductase deficiency 25 NDUFAF1 Isolated NADH-CoQ reductase deficiency 25 NDUFAF2 Isolated NADH-CoQ reductase deficiency 25 NDUFAF3 Isolated NADH-CoQ reductase deficiency 25 NDUFAF4 Isolated NADH-CoQ reductase deficiency 25 NDUFAF5 Isolated NADH-CoQ reductase deficiency . 25 NDUFB3 Isolated NADH-CoQ reductase deficiency 25 NDUFB9 Isolated NADH-CoQ reductase deficiency 25 NDUFS1 Leigh syndrome with leukodystrophy 13 NDUFS1 Isolated NADH-CoQ reductase deficiency 25 NDUFS2 Isolated NADH-CoQ reductase deficiency 25 NDUFS3 Leigh syndrome with leukodystrophy 13 NDUFS3 Isolated NADH-CoQ reductase deficiency 25 NDUFS4 Leigh syndrome with leukodystrophy 13 NDUFS4 Isolated NADH-CoQ reductase deficiency 25 NDUFS6 Isolated NADH-CoQ reductase deficiency 25 NDUFS7 Leigh syndrome with leukodystrophy 13 NDUFS7 Isolated NADH-CoQ reductase deficiency 25 NDUFS8 Leigh syndrome with leukodystrophy . 13 NDUFS8 Isolated NADH-CoQ reductase deficiency 25 NDUFV1 Leigh syndrome with leukodystrophy 13 NDUFV1

Table IX-21781338.1 Isolated NADH-CoQ reductase deficiency 25 NDUFV2 Childhood-onset nemaline myopathy 5 NEB Intermediate nemaline myopathy 3 NEB Severe congenital nemaline myopathy 3 NEB Typical nemaline myopathy 4 NEB Amyotrophic lateral sclerosis 23 NEFH Autosomal dominant Charcot-Marie-Tooth disease type 2 E 1 NEFL Charcot-Marie-Tooth disease type I F 1 NEFL Severe early-onset axonal neuropathy due to NEFL deficiency 1 NEFL Severe early-onset axonal neuropathy due to NEFL deficiency 1 NEFL Short rib-polydactyly syndrome, Majewski type 2 NEK1 Infantile autosomal recessive medullary cystic kidney disease 3 NEK8 Renal-hepatic-pancreatic dysplasia 2 NEK8 Congenital sialidosis type 2 1 NEU1 Sialidosis type 1 1 NEU1 MODY syndrome 12 NEUROD1 Congenital malabsorptive diarrhea due t o paucity of enteroendocrine cells 1 NEUROG3 Familial isolated dilated cardiomyopathy 38 NEXN 17qll microdeletion syndrome 3 NF1 17qll.2 microduplication syndrome 1 NF1 Familial segmental neurofibromatosis 1 NF1 Familial spinal neurofibromatosis 1 NF1 Juvenile myelomonocytic leukemia 3 NF1 Neurofibromatosis type 1 1 NF1 Noonan syndrome 10 NF1 Watson syndrome 1 NF1 Neurofibromatosis type 2 1 NF2 Neurofibromatosis type 3 2 NF2 Marshall-Smith syndrome 1 NFIX Sotos syndrome 2 NFIX Giant cell glioblastoma 10 NFKBIA Gliosarcoma 10 NFKBIA Hypohidrotic ectodermal dysplasia with immunodeficiency 2 NFKBIA Fatal infantile encephalopathy-pulmonary hypertension syndrome 1 NFU1 Fatal multiple mitochondrial dysfunction syndrome 2 NFU1 Hereditary sensory and autonomic neuropathy type 5 2 NGF

Bilateral generalized polymicrogyria 2 NH EJ 1

Cernunnos-XLF deficiency 1 NH EJ 1 Lafora disease 2 NHLRC1 Dyskeratosis congenita 10 NHP2 Nance-Horan syndrome 1 NHS

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Table IX-21781338.1 Nuclear cataract 9 NHS Total congenital cataract 5 NHS Microcephalic primordial dwarfism, Dauber type 1 NIN Autosomal dominant spastic paraplegia type 6 1 NIPA1 Congenital nonbullous ichthyosiform erythroderma 6 NIPAL4 Lamellar ichthyosis 6 NIPAL4 5pl3 microduplication syndrome 1 NIPBL Cornelia de Lange syndrome 5 NIPBL Athyreosis 5 NKX2-1 Benign familial chorea 1 NKX2-1 Brain-lung-thyroid syndrome 1 NKX2-1 Papillary or follicular thyroid carcinoma 14 NKX2-1 Thyroid hypoplasia 5 NKX2-1 Athyreosis 5 NKX2-5 Atrial septal defect - atrioventricular conduction defects 1 NKX2-5 Atrial septal defect, ostium secundum type 8 NKX2-5 Familial atrial fibrillation 14 NKX2-5 Familial isolated congenital asplenia 2 NKX2-5 Familial progressive cardiac conduction defect 3 NKX2-5 Hypoplastic left heart syndrome 2 NKX2-5 Tetralogy of Fallot 8 NKX2-5 Thyroid hypoplasia 5 NKX2-5 Ventricular septal defect 4 NKX2-5 Truncus arteriosus 1 NKX2-6 Spondylo-megaepiphyseal-metaphyseal dysplasia 1 NKX3-2 Corneal intraepithelial dyskeratosis with palmoplantar hyperkeratosis and laryngeal dyskeratosis 1 NLRP1 Vitiligo-associated autoimmune disease 2 NLRP1 NLRP12-associated hereditary periodic fever syndrome 1 NLRP12 CINCA syndrome with CIAS1 mutations 1 NLRP3 Familial cold urticaria 1 NLRP3 Muckle-Wells syndrome 1 NLRP3 Complete hydatidiform mole 2 NLRP7 Partial hydatidiform mole 1 NLRP7 Primary ciliary dyskinesia 21 NME8 Leber congenital amaurosis 18 NM NAT1 Familial glucocorticoid deficiency 4 NNT Behcet disease 9 NOD2 Blau syndrome 1 NOD2 Alobar holoprosencephaly 14 NODAL Lobar holoprosencephaly 14 NODAL

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Table IX-21781338.1 Microform holoprosencephaly 14 NODAL Midline interhemispheric variant of holoprosencephaly 14 NODAL Semilobar holoprosencephaly 14 NODAL Septopreoptic holoprosencephaly 14 NODAL Situs ambiguus 6 NODAL Situs inversus totalis 3 NODAL Brachydactyly type B2 1 NOG Multiple synostoses syndrome 3 NOG Proximal symphalangism 2 NOG Stapes ankylosis with broad thumbs and toes 1 NOG Tarsal-carpal coalition syndrome 1 NOG Familial cortical myoclonus 1 NOL3 Translocation renal cell carcinoma 7 NONO Dyskeratosis congenita 10 NOP10 Spinocerebellar ataxia type 36 1 NOP56 Romano-Ward syndrome 13 NOS1AP Acroosteolysis dominant type 1 NOTCH2 Alagille syndrome due to a NOTCH2 point mutation 1 NOTCH2 CADASI L 1 NOTCH3 Niemann-Pick disease type C, adult neurologic onset 2 NPC1 Niemann-Pick disease type C, juvenile neurologic onset 2 NPC1 Niemann-Pick disease type C, late infantile neurologic onset 2 NPC1 Niemann-Pick disease type C, severe early infantile neurologic onset 2 NPC1 Niemann-Pick disease type C, severe perinatal 2 NPC1 Niemann-Pick disease type C, adult neurologic onset 2 NPC2 Niemann-Pick disease type C, juvenile neurologic onset 2 NPC2 Niemann-Pick disease type C, late infantile neurologic onset 2 NPC2 Niemann-Pick disease type C, severe early infantile neurologic onset 2 NPC2 Niemann-Pick disease type C, severe perinatal 2 NPC2 Joubert syndrome with renal defect 5 NPH P1 Juvenile autosomal recessive medullary cystic kidney disease 4 NPHP1 Senior-Loken syndrome 8 NPHP1 Late-onset autosomal recessive medullary cystic kidney disease 3 NPHP3 NPH P3-related Meckel-like syndrome 1 NPHP3 Renal-hepatic-pancreatic dysplasia 2 NPHP3 Senior-Loken syndrome 8 NPHP3 Juvenile autosomal recessive medullary cystic kidney disease 4 NPHP4 Senior-Loken syndrome 8 NPHP4 Congenital nephrotic syndrome, Finnish type 1 NPHS1 Familial idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial proliferation 2 NPHS1

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Table IX-21781338.1 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 NPHS1 Familial idiopathic steroid-resistant nephrotic syndrome with minimal changes 3 NPHS1 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 NPHS2 Familial idiopathic steroid-resistant nephrotic syndrome with minimal changes 3 NPHS2 Sporadic idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 2 NPHS2 Sporadic idiopathic steroid-resistant nephrotic syndrome with minimal changes 1 NPHS2 Acute promyelocytic leukemia 4 NPM1 Familial atrial fibrillation 14 NPPA Acromesomelic dysplasia, Maroteaux type 1 NPR2 Tall stature - scoliosis - macrodactyly of the great toes 1 NPR2 46,XY complete gonadal dysgenesis 8 NR0B1 46,XY partial gonadal dysgenesis 7 NR0B1 Cytomegalic congenital adrenal hypoplasia 1 NR0B1 Amyotrophic lateral sclerosis 23 NR1H3 Intrahepatic cholestasis of pregnancy 4 NR1H4 Goldmann-Favre syndrome 1 NR2E3 Retinitis pigmentosa 6 1 NR2E3 ' Glucocorticoid resistance 1 NR3C1 Glucocorticoid resistance 1 NR3C1 Pseudohyperaldosteronism type 2 1 NR3C2 Renal pseudohypoaldosteronism type 1 1 NR3C2 Young adult-onset Parkinsonism 13 NR4A2 46,XX gonadal dysgenesis 4 NR5A1 46 XY complete gonadal dysgenesis 8 NR5A1 46,XY disorder of sex development - adrenal insufficiency due to CYP11A1 deficiency 2 NR5A1 46,XY partial gonadal dysgenesis 7 NR5A1 Autoimmune lymphoproliferative syndrome 6 NRAS Noonan syndrome 10 NRAS Retinitis pigmentosa 61 NRL Hirschsprung disease 7 NRTN Pitt-Hopkins-like syndrome 2 NRXN1 5q35 microduplication syndrome 1 NSD1 5q35 microduplication syndrome 1 NSD1 Beckwith-Wiedemann syndrome due t o NSD1 mutation 1 NSD1 . Sotos syndrome 2 NSD1 Weaver syndrome 2 NSD1 138

Table IX-21781338.1 CHILD syndrome 1 NSDHL CK syndrome 1 NSDHL Kallmann syndrome 19 NSMF Normosmic congenital hypogonadotropic hypogonadism 18 NSMF Autosomal recessive nonsyndromic intellectual deficit 15 NSUN2 Du bowitz syndrome 1 NSUN2 Hemolytic anemia due to pyrimidine 5' nucleotidase deficiency 1 NT5C3 Hereditary arterial and articular multiple calcification syndrome 1 NT5E Hereditary arterial and articular multiple calcification syndrome 1 NT5E Atypical Rett syndrome 4 NTNG1 Familial medullary thyroid carcinoma 2 NTRK1 Hereditary sensory and autonomic neuropathy type 4 1 NTRK1 Hereditary sensory and autonomic neuropathy type 5 2 NTRK1 Papillary or follicular thyroid carcinoma 14 NTRK1 Congenital mesoblastic nephroma 2 NTRK3 Fibrosarcoma 2 NTRK3 Isolated NADH-CoQ reductase deficiency 25 NUBPL Familial atrial fibrillation 14 NUP155 Familial infantile bilateral striatal necrosis 2 NUP62 Familial infantile bilateral striatal necrosis 2 NUP62 Congenital stationary night blindness 12 NYX Gyrate atrophy of choroid and retina 1 OAT Idiopathic pulmonary fibrosis 11 OBFC1 3 syndrome 3 OBSL1 Angelman syndrome 6 OCA2 Oculocutaneous albinism type 2 2 OCA2 Prader-Willi syndrome 6 OCA2 Congenital intrauterine infection-like syndrome 1 OCLN Dent disease type 2 1 OCRL Oculocerebrorenal syndrome 1 OCRL Colobomatous microphthalmia 8 ODZ3 Joubert syndrome with orofaciodigital defect 4 OFD1 Oral-facial-digital syndrome type 1 1 OFD1 Primary ciliary dyskinesia 21 OFD1 Retinitis pigmentosa 61 OFD1 Simpson-Golabi-Behmel syndrome type 2 1 O.FD1 Oxoglutaricaciduria 1 OGDH Autosomal dominant optic atrophy and congenital deafness 1 OPA1 Autosomal dominant optic atrophy plus syndrome 2 OPA1 Autosomal dominant optic atrophy, classic type 1 OPA1 Early-onset X-linked optic atrophy 1 OPA2

Table IX-21781338.1 3-methylglutaconic aciduria type 3 2 OPA3 Autosomal dominant optic atrophy and cataract 1 OPA3 X-linked intellectual deficit - cerebellar hypoplasia 1 OPHN1 5-oxoprolinase deficiency 1 OPLAH Cone rod dystrophy 22 OPN1LW Blue cone monochromatism 2 OPN1MW Cone rod dystrophy 22 OPN1MW Tritanopia 1 OPN1SW Amyotrophic lateral sclerosis 23 OPTN Combined immunodeficiency due to O AI1 deficiency 1 ORAI1 Combined immunodeficiency due t o ORAI1 deficiency 1 ORAI1 Ear-patella-short stature syndrome 5 ORC1 Ear-patella-short stature syndrome 5 ORC4 Ear-patella-short stature syndrome 5 ORC6 Familial primary localized cutaneous amyloidosis 2 OSMR Infantile osteopetrosis with neuroaxonal dysplasia 1 OSTM1 Ornithine transcarbamylase deficiency 1 OTC Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOA Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOF Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOG Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 OTOGL Agnathia - holoprosencephaly - situs inversus 2 OTX2 Combined pituitary hormone deficiencies, genetic forms 5 OTX2 Septo-optic dysplasia 6 OTX2 Syndromic microphthalmia type 5 1 OTX2 Succinyl-CoA acetoacetate transferase deficiency 1 OXCT1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 P2RX2 P2Y12 deficiency 1 P2RY12 Oculopharyngeal muscular dystrophy 1 PABPN1 Young adult-onset Parkinsonism 13 PACRG Intellectual deficit - craniofacial dysmorphism - cryptorchidism 1 PACS1 17pl3.3 microduplication syndrome 2 PAFAH1B1 Lissencephaly due t o LIS1 mutation 1 PAFAH1B1 Miller-Dieker syndrome 3 PAFAH1B1 Subcortical band heterotopia 2 PAFAH1B1 Maternal hyperphenylalaninemia 1 PAH Mild hyperphenylalaninemia 1 PAH Mild phenylketonuria 1 PAH Tetrahydrobiopterin-responsive hyperphenylalaninemia/phenylketonuria 1 PAH Tetrahydrobiopterin-responsive hyperphenylalaninemia/phenylketonuria 1 PAH X-linked nonsyndromic intellectual deficit 24 PAK3

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Table IX-21781338.1 Familial pancreatic carcinoma 8 PALB2 Fanconi anemia 16 PALB2 Hereditary breast and ovarian cancer syndrome 13 PALB2 Atypical pantothenate kinase associated neurodegeneration 1 PANK2 Classic pantothenate kinase associated neurodegeneration 1 PANK2 Brachyolmia type 1, Toledo type 1 PAPSS2 Spondyloepimetaphyseal dysplasia, Pakistani type 1 PAPSS2 Young adult-onset Parkinsonism 13 PARK2 Amyotrophic lateral sclerosis-parkinsonism-dementia complex 2 PARK7 Young adult-onset Parkinsonism 13 PARK7 Renal coloboma syndrome 1 PAX2 Alveolar rhabdomyosarcoma 4 PAX3 Craniofacial-deafness-hand syndrome 1 PAX3 Waardenburg syndrome type 1 1 PAX3 Waardenburg syndrome type 3 1 PAX3 MODY syndrome 12 PAX4 Aniridia - cerebellar ataxia - intellectual deficit 1 PAX6 Autosomal dominant keratitis 1 PAX6 Foveal hypoplasia - presenile cataract 1 PAX6 Isolated aniridia 1 PAX6 Isolated aniridia 1 PAX6 Isolated optic nerve hypoplasia 1 PAX6 Morning glory syndrome 1 PAX6 Peters anomaly 6 PAX6 W AG syndrome 3 PAX6 Alveolar rhabdomyosarcoma 4 PAX7 Athyreosis 5 PAX8 Thyroid hypoplasia 5 PAX8 Oligodontia 10 PAX9 Precursor B-cell acute lymphoblastic leukemia 13 PBX1 Pyruvate carboxylase deficiency, benign type 1 PC Pyruvate carboxylase deficiency, infantile type 1 PC Pyruvate carboxylase deficiency, severe neonatal type 1 PC Dehydratase deficiency 1 PCBD1 Propionic acidemia 2 PCCA Propionic acidemia 2 PCCB Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 PCDH15 Usher syndrome type 1 9 PCDH15 Female restricted epilepsy with intellectual deficit 1 PCDH19 Phosphoenolpyruvate carboxykinase 1 deficiency 1 PCK1 Papillary or follicular thyroid carcinoma 14 PCM1

Table IX-21781338.1 Microcephalic osteodysplastic primordial dwarfism type 2 1 PCNT Seckel syndrome 6 PCNT Obesity due t o prohormone convertase-l deficiency 1 PCSK1 Hereditary cerebral cavernous malformation 3 PDCD10 Primary pigmented nodular adrenocortical disease 3 PDE11A Acrodysostosis 2 PDE4D Acrodysostosis with multiple hormone resistance 2 PDE4D Retinitis pigmentosa 61 PDE6A Congenital stationary night blindness 12 PDE6B Retinitis pigmentosa 61 PDE6B Achromatopsia 6 PDE6C Progressive cone dystrophy 4 PDE6C Retinitis pigmentosa 61 PDE6G Achromatopsia 6 PDE6H Autosomal dominant striatal neurodegeneration 1 PDE8B Primary pigmented nodular adrenocortical disease 3 PDE8B Dermatofibrosarcoma protuberans 2 PDGFB Familial multiple meningioma 5 PDGFB Gastrointestinal stromal tumor 4 PDGFRA Idiopathic hypereosinophilic syndrome 3 PDGFRA Myeloid neoplasm associated with PDGFRA rearrangement 1 PDGFRA Precursor B-cell acute lymphoblastic leukemia 13 PDGFRA Bilateral striopallidodentate calcinosis 2 PDGFRB Chronic myelomonocytic leukemia PDGFRB Idiopathic hypereosinophilic syndrome 3 PDGFRB Infantile myofibromatosis 1 PDGFRB Myeloid neoplasm associated with PDGFRB rearrangement 1 PDGFRB Unclassified chronic myeloproliferative disease 1 PDGFRB Leigh syndrome with cardiomyopathy 8 PDHA1 Pyruvate dehydrogenase El-alpha deficiency 1 PDHA1 Pyruvate dehydrogenase El-beta deficiency 1 PDH B Pyruvate dehydrogenase E3-binding protein deficiency 1 PDHX X-linked Charcot-Marie-Tooth disease type 6 1 PDK3 Pyruvate dehydrogenase phosphatase deficiency 1 PDP1 Deafness - encephaloneuropathy - obesity - valvulopathy 1 PDSS1 Leigh syndrome with nephrotic syndrome 4 PDSS2 MODY syndrome 12 PDX1 Partial pancreatic agenesis 1 PDX1 Permanent neonatal diabetes mellitus 5 PDX1 Spinocerebellar ataxia type 23 1 PDYN Usher syndrome type 2 4 PDZD7

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Table IX-21781338.1 Prolidase deficiency 1 PEPD Familial advanced sleep-phase syndrome 2 PER2 Infantile efsum disease 13 PEX1 Neonatal adrenoleukodystrophy 13 PEX1 Zellweger syndrome 13 PEX1 Autosomal recessive ataxia due t o PEXIO deficiency 1 PEXIO Infantile Refsum disease 13 PEXIO Neonatal adrenoleukodystrophy 13 PEXIO Zellweger syndrome 13 PEXIO Infantile Refsum disease 13 PEX11B Neonatal adrenoleukodystrophy 13 PEX11B Zellweger syndrome 13 PEX11B Infantile Refsum disease 13 PEX12 Neonatal adrenoleukodystrophy 13 PEX12 Zellweger syndrome 13 PEX12 Infantile Refsum disease 13 PEX13 Neonatal adrenoleukodystrophy 13 PEX13 Zellweger syndrome 13 PEX13 Infantile Refsum disease 13 PEX14 Neonatal adrenoleukodystrophy 13 PEX14 Zellweger syndrome 13 PEX14 Infantile Refsum disease 13 PEX16 Neonatal adrenoleukodystrophy 13 PEX16 Zellweger syndrome 13 PEX16 Infantile Refsum disease 13 PEX19 Neonatal adrenoleukodystrophy 13 PEX19 Zellweger syndrome 13 PEX19 Infantile Refsum disease 13 PEX2 Neonatal adrenoleukodystrophy 13 PEX2 Zellweger syndrome 13 PEX2 Infantile Refsum disease 13 PEX26 Neonatal adrenoleukodystrophy 13 PEX26 Zellweger syndrome 13 PEX26 Infantile Refsum disease 13 PEX3 Neonatal adrenoleukodystrophy 13 PEX3 Zellweger syndrome 13 PEX3 Infantile Refsum disease 13 PEX5 Neonatal adrenoleukodystrophy 13 PEX5 Zellweger syndrome 13 PEX5 Infantile Refsum disease 13 PEX6 Neonatal adrenoleukodystrophy 13 PEX6

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Table IX-21781338.1 Zellweger syndrome 13 PEX6 Refsum disease 2 PEX7 Rhizomelic chondrodysplasia punctata type 1 1 PEX7 Glycogen storage disease due to muscle phosphofructokinase deficiency 1 PFKM Amyotrophic lateral sclerosis 23 PFN 1 Glycogen storage disease due to phosphoglycerate mutase deficiency 1 PGAM2 Hyperphosphatasia-intellectual deficiency syndrome 3 PGAP2 Glycogen storage disease due to phosphoglycerate kinase 1 deficiency 1 PGK1 Glycogen storage disease due to phosphoglucomutase deficiency 1 PGM1 PGM-CDG syndrome 1 PGM1 Autosomal recessive primary microcephaly 11 PHC1 X-linked hypophosphatemia 1 PHEX Potocki-Shaffer syndrome 3 PHF21A Borjeson-Forssman-Lehmann syndrome 1 PHF6 Intellectual deficit, X-linked, Siderius type 1 PHF8 3-Phosphoglycerate dehydrogenase deficiency 1 PHGDH Glycogen storage disease due to muscle phosphorylase kinase deficiency PHKA1 Glycogen storage disease due to liver phosphorylase kinase deficiency 2 PHKA2 Glycogen storage disease due to liver and muscle phosphorylase kinase deficiency 1 PHKB Glycogen storage disease due to muscle phosphorylase kinase deficiency 2 PHKG1 Glycogen storage disease due to liver phosphorylase kinase deficiency 2 PHKG2 Congenital fibrosis of extraocular muscles 4 PHOX2A Haddad syndrome 3 PHOX2B Neuroblastoma 7 PHOX2B Ondine syndrome 5 PHOX2B Refsum disease 2 PHYH Dehydrated hereditary stomatocytosis 1 PIEZOl Arthrogryposis with oculomotor limitation and electroretinal anomalies 1 PIEZ02 Multiple congenital anomalies-hypotonia-seizures syndrome type 2 1 PIGA Paroxysmal nocturnal hemoglobinuria 1 PIGA Zunich-Kaye syndrome 1 PIGL Hypercoagulability syndrome due t o glycosylphosphatidylinositol deficiency ' 1 PIGM Multiple congenital anomalies - hypotonia - seizures syndrome 1 PIGN Hyperphosphatasia-intellectual deficiency syndrome 3 PIGO Hyperphosphatasia-intellectual deficiency syndrome 3 PIGV CLOVE syndrome 1 PIK3CA Cowden syndrome 7 PIK3CA Hemimegalencephaly 2 PIK3CA Hereditary nonpolyposis colon cancer 11 PIK3CA

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Table IX-21781338.1 Macrocephaly-capillary malformation syndrome 1 PIK3CA Macrodactyly of fingers, unilateral 1 PIK3CA Segmental progressive overgrowth syndrome with fibroadipose hyperplasia 1 PIK3CA Autosomal agammaglobulinemia 7 PIK3R1 Megalencephaly - polymicrogyria - post-axial Polydactyly - hydrocephalus 2 PIK3R2 Spinocerebellar ataxia with axonal neuropathy type 2 2 PIK3R5 Fleck corneal dystrophy 1 PIKFYVE Young adult-onset Parkinsonism 13 PINK1 Lethal congenital contracture syndrome type 3 2 PIP5K1C Cone rod dystrophy 22 PITPNM3 Brachydactyly - elbow wrist dysplasia 1 PITX1 Familial clubfoot due t o 5q31 microdeletion 1 PITX1 Familial clubfoot due t o PITX1 point mutation 1 PITX1 Axenfeld-Rieger syndrome 2 PITX2 Axenfeld's anomaly 2 PITX2 Peters anomaly 6 PITX2 Rieger's anomaly 2 PITX2 Ring dermoid of cornea l ' PITX2 Cataract-glaucoma 1 PITX3 Familial ocular anterior segment mesenchymal dysgenesis 2 PITX3 Posterior polar cataract 5 PITX3 Autosomal dominant polycystic kidney disease type 1 with tuberous sclerosis 2 PKD1 Autosomal recessive polycystic kidney disease 1 PKHD1 Hemolytic anemia due t o red cell pyruvate kinase deficiency 1 PKLR Epidermolysis bullosa simplex due t o plakophilin deficiency 1 PKP1 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 PKP2 Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 PKP2 Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 PKP2 Adult-onset dystonia-parkinsonism 1 PLA2G6 Infantile neuroaxonal dystrophy 1 PLA2G6 Paternal uniparental disomy of chromosome 6 2 PLAGL1 Transient neonatal diabetes mellitus 5 PLAGLl Quebec platelet disorder 1 PLAU Early infantile epileptic encephalopathy 9 PLCB1 Malignant migrating partial seizures of infancy 4 PLCB1 Auriculo-condylar syndrome 2 PLCB4 Leukonychia totalis 1 PLCD1 Familial idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial sclerosis 4 PLCE1

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Table IX-21781338.1 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 PLCE1 Autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation 1 PLCG2 PLCG2-associated antibody deficiency and immune dysregulation 1 PLCG2 Hermansky-Pudlak syndrome type 9 1 PLDN Autosomal recessive limb-girdle muscular dystrophy type 2Q 1 PLEC Epidermolysis bullosa simplex with muscular dystrophy 1 PLEC Epidermolysis bullosa simplex with pyloric atresia 2 PLEC Epidermolysis bullosa simplex, Ogna type 1 PLEC Spinocerebellar ataxia type 4 1 PLEKHG4 Autosomal recessive lower motor neuron disease with childhood onset 1 PLEKHG5 Intermediate osteopetrosis 2 PLEKHM1 Hypoplasminogenemia 1 PLG Ligneous conjunctivitis 1 PLG Familial partial lipodystrophy associated with PLIN1 mutations 1 PLINl Familial isolated dilated cardiomyopathy 38 PLN Ehlers-Danlos syndrome, kyphoscoliotic type , 1 PLOD1 Nevo syndrome 1 PLOD1 Bruck syndrome 2 PLOD2 Connective tissue disorder due t o lysyl hydroxylase-3 deficiency 1 PLOD3 Null syndrome 1 PLP1 Pelizaeus-Merzbacher disease in female carriers 1 PLP1 Pelizaeus-Merzbacher disease, classic form 1 PLP1 Pelizaeus-Merzbacher disease, connatal form 1 PLP1 Pelizaeus-Merzbacher disease, transitional form 1 PLP1 Spastic paraplegia type 2 1 PLP1 Acute promyelocytic leukemia 4 PML PMM2-CDG syndrome 1 PMM2 . Acute inflammatory demyelinating polyradiculoneuropathy 1 PMP22 Charcot-Marie-Tooth disease type 1A 1 PMP22 Charcot-Marie-Tooth disease type I E 1 PMP22 Dejerine-Sottas syndrome 4 PMP22 Hereditary neuropathy with liability t o pressure palsies 1 PMP22 Roussy-Levy syndrome 2 PMP22 Hereditary nonpolyposis colon cancer 11 PMS1 Constitutional mismatch repair deficiency syndrome 4 PMS2 Hereditary nonpolyposis colon cancer 11 PMS2 Nonpolyposis Turcot syndrome 4 PMS2 Paroxysmal non-kinesigenic dyskinesia 1 PNKD Microcephaly - seizures - developmental delay 1 PNKP

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Table IX-21781338.1 Purine nucleoside phosphorylase deficiency 1 PNP Congenital nonbullous ichthyosiform erythroderma 6 PNPLA1 Neutral lipid storage myopathy 1 PNPLA2 Autosomal recessive spastic paraplegia type 39 1 PNPLA6 Pyridoxal phosphate-responsive seizures 1 PNPO Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 PNPT1 Combined oxidative phosphorylation defect type 13 1 PNPT1 Short stature-onychodysplasia-facial dysmorphism-hypotrichosis syndrome 1 POC1A Dowling-Degos disease 2 POFUT1 Facial dysmorphism - immunodeficiency - livedo - short stature 1 POLE Alpers syndrome 1 POLG Autosomal dominant progressive external ophthalmoplegia 5 POLG Autosomal recessive progressive external ophthalmoplegia 2 POLG Mitochondrial neurogastrointestinal encephalomyopathy 3 POLG Recessive mitochondrial ataxic syndrome 1 POLG Sensory ataxic neuropathy - dysarthria - ophthalmoparesis 2 POLG Spinocerebellar ataxia with epilepsy 1 POLG Autosomal dominant progressive external ophthalmoplegia 5 POLG 2 Xeroderma pigmentosum variant 1 POLH Treacher-Collins syndrome 3 POLR1C Treacher-Collins syndrome 3 POLR1D Hypomyelination - hypogonadotropic hypogonadism - hypodontia 2 POLR3A Odontoleukodystrophy 1 POLR3A Hypomyelination - hypogonadotropic hypogonadism - hypodontia 2 POLR3B Obesity due to pro-opiomelanocortin deficiency 1 POMC Autosomal recessive limb-girdle muscular dystrophy type 20 1 POMGNT1 Muscle eye brain disease 7 POMGNT1 Walker-Warburg syndrome · 13 POMGNT1 Keratosis linearis-ichthyosis congenita-sclerosing keratoderma syndrome 1 POMP Autosomal recessive limb-girdle muscular dystrophy type 2 K 1 POMT1 Congenital muscular dystrophy - muscle hypertrophy - severe intellectual deficit 1 POMT1 Muscle eye brain disease 7 POMT1 Walker-Warburg syndrome 13 POMT1 Autosomal recessive limb-girdle muscular dystrophy type 2N 1 POMT2 Muscle eye brain disease 7 POMT2 Walker-Warburg syndrome 13 POMT2 Amyotrophic lateral sclerosis 23 PON1 Amyotrophic lateral sclerosis 23 PON2 Amyotrophic lateral sclerosis 23 PON3

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Table IX-21781338.1 Anauxetic dysplasia 2 POP1 Congenital adrenal hyperplasia due t o cytochrome P450 oxidoreductase deficiency 1 POR Focal dermal hypoplasia 1 PORCN B-cell chronic lymphocytic leukemia 7 POT1 Combined pituitary hormone deficiencies, genetic forms 5 POU1F1 Hypothyroidism due to deficient transcription factors involved in pituitary development or function 5 POU1F1 Primary biliary cirrhosis 8 POU2AF1 Developmental delay - deafness, Hildebrand type 1 POU3F4 Gusher syndrome 1 POU3F4 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 POU4F3 Nephroblastoma 5 POU6F2 Familial partial lipodystrophy associated with PPARG mutations 1 PPARG Giant cell glioblastoma 10 PPARG Gliosarcoma 10 PPARG Osteogenesis imperfecta type 2 5 PPIB Osteogenesis imperfecta type 3 9 PPIB Osteogenesis imperfecta type 4 7 PPIB 2p21 microdeletion syndrome 4 PPM1B Intermediate maple syrup urine disease 5 PPM 1K Porphyria variegata 2 PPOX Spinocerebellar ataxia type 12 1 PPP2R2B CLN 1 disease 1 PPT1 Hamel cerebro-palato-cardiac syndrome 1 PQBP1 X-linked intellectual deficit, Porteous type 1 PQBP1 X-linked intellectual deficit, Sutherland-Haan type 1 PQBP1 Translocation renal cell carcinoma 7 PRCC Retinitis pigmentosa 61 PRCD lp36 deletion syndrome 4 PRDM16 Familial isolated dilated cardiomyopathy 38 PRDM16 Left ventricular noncompaction 11 PRDM16 Brittle cornea syndrome 2 PRDM5 Early-onset Lafora body disease 1 PRDM8 2p21 microdeletion syndrome 4 PREPL Atypical hypotonia - cystinuria syndrome 3 PREPL Hypotonia - cystinuria syndrome 2 PREPL Familial hemophagocytic lymphdhistiocytosis 4 PRF1 Idiopathic aplastic anemia 5 PRF1 Camptodactyly-arthropathy-coxa-vara-pericarditis syndrome 1 PRG4 Unverricht-Lundborg disease 3 PRICKLEl

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Table IX-21781338.1 Acrodysostosis 2 PRKAR1A Acrodysostosis with multiple hormone resistance 2 PRKAR1A Carney complex 1 PRKAR1A Familial atrial myxoma 1 PRKAR1A Primary pigmented nodular adrenocortical disease 3 PRKAR1A Autoimmune lymphoproliferative syndrome 6 PRKCD Common variable immunodeficiency 10 PRKCD Spinocerebellar ataxia type 14 1 PRKCG Isolated polycystic liver disease 2 PRKCSH Severe combined immunodeficiency due t o DNA-PKcs deficiency 1 PRKDC Dystonia 16 1 PRKRA Familial Alzheimer-like prion disease 1 PRNP Fatal familial insomnia 1 PRNP Gerstmann-Straussler-Scheinker syndrome 1 PRNP Gerstmann-Straussler-Scheinker syndrome 1 PRNP Huntington disease-like 1 1 PRNP Inherited Creutzfeldt-Jakob disease 1 PRNP Hereditary thrombophilia due t o congenital protein C deficiency 1 PROC Hyperprolinemia type 1 1 PRODH Kallmann syndrome 19 PROK2 Normosmic congenital hypogonadotropic hypogonadism 18 PROK2 Kallmann syndrome 19 PROKR2 Normosmic congenital hypogonadotropic hypogonadism 18 PROKR2 Septo-optic dysplasia 6 PROKR2 Cone rod dystrophy 22 PROM1 Retinal macular dystrophy type 2 1 PROM1 Retinitis pigmentosa 6 1 PROM1 Stargardt disease 3 PROM1 Combined pituitary hormone deficiencies, genetic forms 5 PROP1 Hypothyroidism due t o deficient transcription factors involved in pituitary development or function 5 PROP1 Panhypopituitarism 2 PROP1 Hereditary thrombophilia due t o congenital protein Sdeficiency 1 PROS1 Retinitis pigmentosa 61 PRPF3 Retinitis pigmentosa 6 1 PRPF31 Retinitis pigmentosa 6 1 PRPF6 Retinitis pigmentosa 6 1 PRPF8 Amyotrophic lateral sclerosis 23 PRPH Adult-onset foveomacular vitelliform dystrophy 2 PRPH2 Butterfly-shaped pigment dystrophy 1 PRPH2 Central areolar choroidal dystrophy 2 PRPH2

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Table IX-21781338.1 Cone rod dystrophy 22 PRPH2 Fundus albipunctatus 3 PRPH2 Retinitis pigmentosa 6 1 PRPH2 Retinitis punctata albescens 4 PRPH2 Lethal ataxia with deafness and optic atrophy 1 PRPS1 Phosphoribosylpyrophosphate synthetase superactivity 1 PRPS1 X-linked Charcot-Marie-Tooth disease type 5 1 PRPS1 X-linked nonsyndromic sensorineural deafness type DFN 2 PRPS1 Benign familial infantile seizures 4 P RT2 Familial o r sporadic hemiplegic migraine 4 PRRT2 ICCA syndrome 1 PRRT2 Paroxysmal kinesigenic dyskinesia 1 P RT2 Agnathia - holoprosencephaly - situs inversus 2 PRRX1 Hereditary chronic pancreatitis 6 PRSS1 Autosomal recessive nonsyndromic intellectual deficit 15 PRSS12 Hereditary chronic pancreatitis 6 PRSS2 Isolated anophthalmia - microphthalmia 5 PRSS56 Wegener granulomatosis 3 PRTN3 Charcot-Marie-Tooth disease type 4 F 1 PRX Dejerine-Sottas syndrome 4 PRX Atypical Gaucher disease due t o saposin C deficiency 1 PSAP Encephalopathy due to prosaposin deficiency 1 PSAP Infantile Krabbe disease 2 PSAP Metachromatic leukodystrophy, adult form 2 PSAP Metachromatic leukodystrophy, juvenile form 2 PSAP Metachromatic leukodystrophy, late infantile form 2 PSAP Phosphoserine aminotransferase deficiency 1 PSAT1 Behavioral variant of frontotemporal dementia 3 PSEN 1 Early-onset autosomal dominant Alzheimer disease 4 PSEN 1 Familial isolated dilated cardiomyopathy 38 PSEN 1 Progressive non-fluent aphasia 3 PSEN 1 Semantic dementia 2 PSEN 1 Early-onset autosomal dominant Alzheimer disease 4 PSEN2 Familial isolated dilated cardiomyopathy 38 PSEN2 CANDLE syndrome 1 PSMB8 JM P syndrome 1 PSMB8 Nakajo-Nishimura syndrome 1 PSMB8 46,XX gonadal dysgenesis 4 PSMC3IP 3-Phosphoserine phosphatase deficiency 1 PSPH Pyogenic arthritis - pyoderma gangrenosum - acne 1 PSTPIP1 Alobar holoprosencephaly 14 PTCH1

150

Table IX-2 7 338.1 Gorlin syndrome 1 PTCH1 Lobar holoprosencephaly 14 PTCH1 Microform holoprosencephaly 14 PTCH1 Midline interhemispheric variant of holoprosencephaly 14 PTCH1 Semilobar holoprosencephaly 14 PTCH1 Septopreoptic holoprosencephaly 14 PTCH1 Commissural facial cleft 2 PTCH2 Bannayan-Riley-Ruvalcaba syndrome 1 PTEN Cowden syndrome 7 PTEN Hereditary breast and ovarian cancer syndrome 13 PTEN Juvenile polyposis of infancy 2 PTEN Lhermitte-Duclos disease 1 PTEN Macrocephaly-autism syndrome 1 PTEN Proteus syndrome 2 PTEN Proteus-like syndrome 1 PTEN Segmental outgrowth - lipomatosis - arteriovenous malformation - epidermal nevus 1 PTEN Squamous cell carcinoma of head and neck 4 PTEN Permanent neonatal diabetes mellitus - pancreatic and cerebellar agenesis 1 PTF1A Familial isolated hypoparathyroidism due t o impaired PTH secretion 2 PTH Chondrodysplasia, Blomstrand type 1 PTH1R Dental ankylosis 1 PTH1R Eiken syndrome 1 PTH1 R Enchondromatosis 3 PTH1R Metaphyseal chondrodysplasia, Jansen type 1 PTH1R Brachydactyly type E 2 PTHLH Juvenile myelomonocytic leukemia 3 PTPN11 LEOPARD syndrome 3 PTPN11 Metachondromatosis 1 PTPN11 Noonan syndrome 10 PTPN11 Juvenile rheumatoid factor-negative polyarthritis 9 PTPN2 Oligoarticular juvenile arthritis 9 PTPN2 Juvenile rheumatoid factor-negative polyarthritis 9 PTPN22 Oligoarticular juvenile arthritis 9 PTPN22 Wegener granulomatosis 3 PTPN22 T-B+ severe combined immunodeficiency due t o CD45 deficiency 1 PTPRC Familial idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial proliferation 2 PTPRO Familial idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial sclerosis 4 PTPRO Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 PTPRO

Table IX-21781338.1 Familial idiopathic steroid-resistant nephrotic syndrome with minimal changes 3 PTPRO Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 PTPRQ Generalized congenital lipodystrophy with myopathy 1 PTRF 6-pyruvoyl-tetrahydropterin synthase deficiency 1 PTS Mitochondrial myopathy and sideroblastic anemia 2 PUS1 Zlotogora-Ogur syndrome 1 PVRL1 Ectodermal dysplasia - syndactyly syndrome 1 PVRL4 Congenital cataract microcornea with corneal opacity 2 PXDN Autosomal recessive cutis laxa type 2B 1 PYCR1 Geroderma osteodysplastica 2 PYCR1 PYCRl-related DeBarsy syndrome 1 PYCR1 Glycogen storage disease due to liver glycogen phosphorylase deficiency 1 PYGL Glycogen storage disease due to liver glycogen phosphorylase deficiency 1 PYGL Glycogen storage disease due to muscle glycogen phosphorylase deficiency 1 PYGM Glycogen storage disease due t o muscle glycogen phosphorylase deficiency 1 PYGM Dihydropteridine reductase deficiency 1 QDPR Micro syndrome 3 RAB18 Carpenter syndrome 2 RAB23 Griscelli disease type 2 1 RAB27A Cone rod dystrophy 22 RAB28 Smith-McCort dysplasia 2 RAB33B X-linked nonsyndromic intellectual deficit 2 RAB39B Cataract - intellectual deficit - hypogonadism 2 RAB3GAP1 Micro syndrome 3 RAB3GAP1 Cataract - intellectual deficit - hypogonadism 2 RAB3GAP2 Micro syndrome 3 RAB3GAP2 Deafness - intellectual deficit, Martin-Probst type 1 RAB40AL Autosomal dominant Charcot-Marie-Tooth disease type 2B 1 RAB7A Neutrophil immunodeficiency syndrome 1 RAC2 Cornelia de Lange syndrome 5 RAD21 Hereditary breast and ovarian cancer syndrome 13 RAD50 Nijmegen breakage syndrome-like disorder 1 RAD50 Familial congenital mirror movements 2 RAD51 Hereditary breast and ovarian cancer syndrome 13 RAD51 Fanconi anemia 16 RAD51C Hereditary breast and ovarian cancer syndrome 13 RAD51C Hereditary breast and ovarian cancer syndrome 13 RAD51D LEOPARD syndrome 3 RAF1 Noonan syndrome 10 RAF1 152

Table IX-21781338.1 Combined immunodeficiency T+ B+ due t o partial RAG1 deficiency 1 RAG1 Combined immunodeficiency with skin granulomas 2 RAG1 Omenn syndrome - 9 RAG1 Severe combined immunodeficiency due t o complete RAGl/2 deficiency 2 RAG1 Combined immunodeficiency with skin granulomas 2 RAG2 Omenn syndrome 9 RAG2 Severe combined immunodeficiency due t o complete RAGl/2 deficiency 2 RAG2 17pll.2 microduplication syndrome 1 RAI 1 Smith-Magenis syndrome 3 RAI 1 Acute necrotizing encephalopathy of childhood 2 RANBP2 Familial acute necrotizing encephalopathy 1 RANBP2 Inflammatory myofibroblastic tumor 6 RANBP2 Fetal akinesia deformation sequence 2 RAPSN Lethal multiple pterygium syndrome 4 RAPSN Postsynaptic congenital myasthenic syndromes 9 RAPSN Acute promye!ocytic leukemia 4 RARA Pontocerebellar hypoplasia type 1 4 RARS2 Pontocerebellar hypoplasia type 6 1 RARS2 Parkes Weber syndrome 1 RASA1 Isolated anophthalmia - microphthalmia 5 RAX Cone rod dystrophy 22 RAX2 Familial retinoblastoma 1 RBI Unilateral retinoblastoma 1 RBI Jawad syndrome 1 RBBP8 Seckel syndrome 6 RBBP8 Autoinflammatory syndrome with pyogenic bacterial infection and amylopectinosis 1 RBCK1 TARP syndrome 1 RBM10 Familial isolated dilated cardiomyopathy 38 RBM20 ANE syndrome 1 RBM28 Thrombocytopenia - absent radius 1 RBM8A Partial chromosome Y deletion 8 RBMY1A1 Retinitis pigmentosa 6 1 RBP3 Progressive retinal dystrophy due t o retinol transport defect 1 RBP4 Adams-Oliver syndrome 4 RBPJ Leber congenital amaurosis 18 RD3 Leber congenital amaurosis 18 RDH12 Retinitis pigmentosa 6 1 RDH12 Fundus albipunctatus 3 RDH5 Retinitis punctata albescens 4 RDH5 Autosomal recessive nonsyndromic sensorineural deafness type DFN B 5 2 RDX

153

Table IX-21781338.1 Baller-Gerold syndrome 1 RECQL4 RAPADILINO syndrome 1 RECQL4 Rothmund-Thomson syndrome type 2 1 RECQL4 Autosomal dominant spastic paraplegia type 31 1 REEP1 Distal hereditary motor neuropathy type 5 3 REEP1 Hereditary chronic pancreatitis 6 REG1A Lissencephaly syndrome, Norman-Roberts type 1 RELN Hyperuricemia - anemia - renal failure 1 REN Renal tubular dysgenesis of genetic origin 4 REN Bilateral renal agenesis 2 RET Bilateral renal dysplasia 1 RET Familial medullary thyroid carcinoma 2 RET Haddad syndrome 3 RET Hirschsprung disease 7 RET Multiple endocrine neoplasia type 2A 1 RET Multiple endocrine neoplasia type 2 B 1 RET Papillary or follicular thyroid carcinoma 14 RET Unilateral renal dysplasia 1 RET Williams syndrome 17 RFC2 RFT1-CDG syndrome 1 RFT1 Immunodeficiency by defective expression of HLA class 2 5 RFX5 Hypoplastic pancreas-intestinal atresia-hypoplastic gallbalder syndrome 1 RFX6 Immunodeficiency by defective expression of HLA class 2 5 RFXANK Immunodeficiency by defective expression of HLA class 2 5 RFXAP Retinitis pigmentosa 61 RGR Bradyopsia 2 RGS9 Bradyopsia 2 RGS9BP Overhydrated hereditary stomatocytosis 1 HAG Rh deficiency syndrome 3 RHAG Paimoplantar keratoderma-esophageal carcinoma syndrome 1 RHBDF2 Rh deficiency syndrome 3 RHCE Rh deficiency syndrome 3 RHD Congenital stationary night blindness 12 RHO Retinitis pigmentosa 6 1 RHO Retinitis punctata albescens 4 RHO T-cell immunodeficiency with epidermodysplasia verruciformis 1 RHOH Cone rod dystrophy 22 RIMS1 MACS syndrome 1 RIN2 Bartsocas-Papas syndrome 1 RIPK4 Noonan syndrome 10 RIT1 Bothnia retinal dystrophy 1 RLBP1

Table IX-21781338.1 Fundus albipunctatus 3 RLBP1 Retinitis pigmentosa 61 RLBP1 Retinitis punctata albescens 4 RLBP1 Combined oxidative phosphorylation defect type 11 1 RMND1 Anauxetic dysplasia 2 RMRP Cartilage-hair hypoplasia 1 RMRP Omenn syndrome 9 RMRP Alveolar rhabdomyosarcoma 4 RMST Aicardi-Goutieres syndrome 6 RNASEH2A Aicardi-Goutieres syndrome 6 RNASEH2B Aicardi-Goutieres syndrome 6 RNASEH2C Familial prostate cancer 14 RNASEL Cystic leukoencephalopathy without megalencephaly 1 R ASET2 17qll microdeletion syndrome 3 RNF135 Overgrowth - macrocephaly - facial dysmorphism 1 RNF135 Familial renal cell carcinoma 6 RNF139 Moyamoya disease 2 RNF213 Cerebellar ataxia - hypogonadism 1 RNF216 Microcephalic osteodysplastic primordial dwarfism types 1 and 3 1 RNU4ATAC Familial vesicoureteral reflux 2 ROB02 Horizontal gaze palsy with progressive scoliosis 1 R0B03 Amelo-cerebro-hypohidrotic syndrome 1 ROGDI Retinitis pigmentosa 61 ROMl Autosomal recessive Robinow syndrome 1 ROR2 Brachydactyly type B 1 ROR2 Retinitis pigmentosa 61 RP1 Occult macular dystrophy 1 RP1L1 Retinitis pigmentosa 61 RP1L1 Retinitis pigmentosa 6 1 RP2 Retinitis pigmentosa 6 1 RP9 Choroideremia 2 RPE65 Leber congenital amaurosis 18 RPE65 Retinitis pigmentosa 61 RPE65 Achromatopsia 6 RPGR Cone rod dystrophy 22 RPGR Primary ciliary dyskinesia 21 RPGR Primary ciliary dyskinesia - retinitis pigmentosa 1 RPGR Retinitis pigmentosa 61 RPGR Cone rod dystrophy 22 RPGRIP1 Leber congenital amaurosis 18 RPGRIP1 Meckel syndrome 13 RPGRIP1

155

Table IX-21781338.1 Joubert syndrome with hepatic defect 3 RPGRIP1L Joubert syndrome with renal defect 5 RPGRIP1L Meckel syndrome 13 RPGRIP1L Blackfan-Diamond anemia 11 RPL11 Hypotrichosis simplex 6 RPL2 1 Blackfan-Diamond anemia 11 RPL26 Blackfan-Diamond anemia 11 RPL35A Blackfan-Diamond anemia 11 RPL5 Blackfan-Diamond anemia 11 RPS10 Myelodysplastic syndrome associated with isolated del(5q) chromosome abnormality 1 RPS14 Blackfan-Diamond anemia 11 RPS17 Blackfan-Diamond anemia 11 RPS19 Blackfan-Diamond anemia 11 RPS24 Blackfan-Diamond anemia 11 RPS26 Coffin-Lowry syndrome 1 RPS6KA3 X-linked nonsyndromic intellectual deficit 24 RPS6KA3 Blackfan-Diamond anemia 11 RPS7 Familial isolated congenital asplenia 2 RPSA Adult-onset chronic progressive external ophthalmoplegia with mitochondrial myopathy 1 RRM2B Autosomal dominant progressive external ophthalmoplegia 5 RRM2B Kearns-Sayre syndrome 3 RRM2B Mitochondrial DNA depletion syndrome, encephalomyopathic form with renal tubulopathy 1 RRM2B Mitochondrial neurogastrointestinal encephalomyopathy 3 RRM2B X-linked retinoschisis 1 RSI Primary ciliary dyskinesia 21 RSPH4A Primary ciliary dyskinesia 21 RSPH9 Palmoplantar keratoderma - XX sex reversal - predisposition to squamous . cell carcinoma 1 RSPOl Congenital anonychia 1 RSP04 Dyskeratosis congenita 10 RTEL1 Hoyeraal-Hreidarsson syndrome 4 RTEL1 Maternal uniparental disomy of chromosome 14 3 RTL1 Paternal uniparental disomy of chromosome 14 3 RTL1 Autosomal dominant spastic paraplegia type 12 1 RTN2 Bilateral generalized polymicrogyria 2 RTTN Acute myeloid leukemia with t(8;21)(q22;q22) translocation 3 RUNX1 Chronic myeloid leukemia 3 RUNX1 Familial platelet syndrome with predisposition t o acute myelogenous leukemia 1 RUNX1

15G

Table IX-21781338.1 Precursor B-cell acute lymphoblastic leukemia 13 RUNX1 Acute myeloid leukemia with t(8;21)(q22;q22) translocation 3 RUNX1T1 Cleidocranial dysplasia 1 RUNX2 Metaphyseal dysplasia - maxillary hypoplasia - brachydacty 1 RUNX2 Autosomal dominant centronuclear myopathy 4 RYR1 Benign Samaritan congenital myopathy 1 RYR1 Central core disease 1 RYR1 Congenital multicore myopathy with external ophthalmoplegia 1 RYR1 King-Denborough syndrome 1 RYR1 Malignant hyperthermia 2 RYR1 Moderate multiminicore disease with hand involvement 1 RYR1 Catecholaminergic polymorphic ventricular tachycardia 4 RYR2 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 RYR2 Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 RYR2 Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 RYR2 Secondary amyloidosis 1 SAA1 Autosomal recessive spastic ataxia of Charlevoix-Saguenay 1 SACS Oguchi disease 2 SAG Retinitis pigmentosa 6 1 SAG Townes-Brocks syndrome 1 SALL1 Acro-renal-ocular syndrome 1 SALL4 IVIC syndrome 1 SALL4 Okihiro syndrome due to 20ql3 microdeletion 1 SALL4 Okihiro syndrome due to a point mutation 1 SALL4 Normocalcemic tumoral calcinosis 1 SAMD9 Aicardi-Goutieres syndrome 6 SAMHD1 Chilblain lupus 2 SAMHD1 Chylomicron retention disease 1 SAR1B Sarcosinemia 1 SARDH Keratosis follicularis spinulosa decalvans 2 SAT1 2q32q33 microdeletion syndrome 1 SATB2 2q33.1 microdeletion syndrome 1 SATB2 Idiopathic aplastic anemia 5 SBDS Shwachman-Diamond syndrome 1 SBDS Charcot-Marie-Tooth disease type 4B2 1 SBF2 Lathosterolosis 1 SC5DL Spinocerebellar ataxia type 20 1 SCA20 Spinocerebellar ataxia type 21 1 SCA21 Spinocerebellar ataxia type 25 1 SCA25 Spinocerebellar ataxia type 26 1 SCA26

157

Table IX-21781338.1 Spinocerebellar ataxia type 30 1 SCA30 Action myoclonus - renal failure syndrome 1 SCARB2 Action myoclonus - renal failure syndrome 1 SCARB2 Gaucher disease type 1 2 SCARB2 Unverricht-Lundborg disease 3 SCARB2 Van den Ende-Gupta syndrome 1 SCARF2 Channelopathy-associated congenital insensitivity to pain 3 SCN10A Paroxysmal extreme pain disorder 3 SCN10A Primary erythermalgia 3 SCN10A Sodium channelopathy-related small fiber neuropathy 2 SCN10A Channelopathy-associated congenital insensitivity t o pain 3 SCN11A Paroxysmal extreme pain disorder 3 SCN11A Primary erythermalgia 3 SCN11A Dravet syndrome 4 SCN1A Epilepsy with myoclonic-astatic seizures 2 SCN1A Familial or sporadic hemiplegic migraine 4 SCN1A Generalized epilepsy with febrile seizures-plus context 6 SCN1A Lennox-Gastaut syndrome 2 SCN1A Malignant migrating partial seizures of infancy 4 SCN1A Brugada syndrome 12 SCN1B Dravet syndrome 4 SCN1B Generalized epilepsy with febrile seizures-plus context 6 SCN1B Benign familial infantile seizures 4 SCN2A Benign familial neonatal-infantile seizures 2 SCN2A Early infantile epileptic encephalopathy 9 SCN2A Generalized epilepsy with febrile seizures-plus context 6 SCN2A Brugada syndrome 12 SCN3B Acetazolamide-responsive myotonia 1 SCN4A Hyperkalemic periodic paralysis 1 SCN4A Hypokalemic periodic paralysis 3 SCN4A Myotonia fluctuans 1 SCN4A Myotonia permanens 1 SCN4A Paramyotonia congenita of Von Eulenburg 1 SCN4A Postsynaptic congenital myasthenic syndromes 9 SCN4A Romano-Ward syndrome 13 SCN4B Atrial stand still 2 SCN5A Brugada syndrome 12 SCN5A Familial atrial fibrillation 14 SCN5A Familial isolated dilated cardiomyopathy 38 SCN5A

Familial progressive cardiac conduction defect f 3 SCN5A Idiopathic ventricular fibrillation, not Brugada type 2 SCN5A

1 8

Table IX-21781338.1 Romano-Ward syndrome 13 SCN5A Sick sinus syndrome 3 SCN5A Early infantile epileptic encephalopathy 9 SCN8A - Channelopathy-associated congenital insensitivity t o pain 3 SCN9A Dravet syndrome 4 SCN9A Erythromelalgia 1 SCN9A Generalized epilepsy with febrile seizures-plus context 6 SCN9A Hereditary sensory and autonomic neuropathy type 2 4 SCN9A Paroxysmal extreme pain disorder 3 SCN9A Primary erythermalgia 3 SCN9A Sodium channelopathy-related small fiber neuropathy 2 SCN9A Generalized pseudohypoaldosteronism type 1 3 SCNN1A Idiopathic bronchiectasis 4 SCNN1A Generalized pseudohypoaldosteronism type 1 3 SCNN1B Idiopathic bronchiectasis 4 SCNN1B Liddle syndrome 2 SCNN1B Generalized pseudohypoaldosteronism type 1 3 SCNN1G Idiopathic bronchiectasis 4 SCNN1G Liddle syndrome 2 SCNN1G Fatal infantile cytochrome C oxidase deficiency 5 SCOl Fatal infantile cytochrome C oxidase deficiency 5 SC02 Leigh syndrome with cardiomyopathy 8 SC02 Leukoencephalopathy - dystonia - motor neuropathy 1 SCP2 Leukoencephalopathy - dystonia - motor neuropathy 1 SCP2 Bardet-Biedl syndrome 17 SDCCAG8 Senior-Loken syndrome 8 SDCCAG8 Familial isolated dilated cardiomyopathy 38 SDHA Hereditary pheochromocytoma-paraganglioma 7 SDHA Isolated succinate-CoQ reductase deficiency 3 SDHA Leigh syndrome with leukodystrophy 13 SDHA Isolated succinate-CoQ reductase deficiency 3 SDHAF1 Hereditary pheochromocytoma-paraganglioma 7 SDHAF2 Carney-Stratakis syndrome 3 SDHB Cowden syndrome 7 SDHB Gastrointestinal stromal tumor 4 SDHB Hereditary pheochromocytoma-paraganglioma 7 SDHB Isolated succinate-CoQ reductase deficiency 3 SDHB Carney-Stratakis syndrome 3 SDHC Cowden syndrome 7 SDHC Gastrointestinal stromal tumor 4 SDHC Hereditary pheochromocytoma-paraganglioma . 7 SDHC

159

Table IX-21781338.1 Carcinoid tumor and carcinoid syndrome 1 SDHD Carney-Stratakis syndrome 3 SDHD Cowden syndrome 7 SDHD Hereditary pheochromocytoma-paraganglioma 7 SDHD Craniolenticulosutural dysplasia 1 SEC23A Congenital dyserythropoietic anemia type 2 1 SEC23B Isolated polycystic liver disease 2 SEC63 Short stature-delayed bone age due t o thyroid hormone metabolism deficiency 1 SECISBP2 Kallmann syndrome 19 SEMA3A CHARGE syndrome 2 SEMA3E Cone rod dystrophy 22 SEMA4A Retinitis pigmentosa 6 1 SEMA4A Monosomy 5p 2 SEMA5A Classic multiminicore myopathy 2 SEPN 1 Congenital fiber-type disproportion myopathy 5 SEPN1 Desmin-related myopathy with Mallory body-like inclusions 1 SEPN1 Rigid spine syndrome 1 SEPN1 Pontocerebellar hypoplasia type 2 4 SEPSECS Progressive cerebello-cerebral atrophy 1 SEPSECS Neuralgic amyotrophy 1 SEPT9 M EGDEL syndrome 1 SERAC1 Alpha-1 antitrypsin deficiency 1 SERPINA1 Hemorrhagic disease due to alpha-1 antitrypsin Pittsburgh mutation 1 SERPINA1 Alpha-l-antichymotrypsin deficiency 1 SERPINA3 Corticosteroid-binding globulin deficiency 1 SERPINA6 Congenital isolated thyroxine-binding globulin deficiency 1 SERPINA7 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 5 2 SERPIN B6 Hereditary thrombophilia due t o congenital antithrombin deficiency 1 SERPINC1 Congenital plasminogen activator inhibitor type 1 deficiency 1 SERPINE1 Osteogenesis imperfecta type 3 9 SERPINF1 Congenital alpha2 antiplasmin deficiency 1 SERPINF2 Hereditary angioedema type 1 1 SERPING1 Immunodeficiency due t o an early component of complement deficiency 8 SERPING1 Osteogenesis imperfecta type 3 9 SERPINH 1 Familial encephalopathy with neuroserpin inclusion bodies 1 SERPINI1 Schinzel-Giedion syndrome 1 SETBP1 Amyotrophic lateral sclerosis type 4 1 SETX Spinocerebellar ataxia with axonal neuropathy type 2 2 SETX Nager syndrome 1 SF3B4 Translocation renal cell carcinoma 7 SFPQ

Table IX-21781338.1 Idiopathic pulmonary fibrosis 11 SFTPA1 Idiopathic pulmonary fibrosis 11 SFTPA2 Congenital pulmonary alveolar proteinosis 5 SFTPB Infant acute respiratory distress syndrome 2 SFTPB Neonatal acute respiratory distress with surfactant metabolism deficiency 2 SFTPB Chronic respiratory distress with surfactant metabolism deficiency 1 SFTPC Congenital pulmonary alveolar proteinosis 5 SFTPC Desquamative interstitial pneumonia 1 SFTPC Idiopathic pulmonary fi brosis 11 SFTPC Infant acute respiratory distress syndrome 2 SFTPC Autosomal recessive limb-girdle muscular dystrophy type 2D 1 SGCA Autosomal recessive limb-girdle muscular dystrophy type 2 E 1 SGCB Autosomal recessive limb-girdle muscular dystrophy type 2 F 1 SGCD Familial isolated dilated cardiomyopathy 38 SGCD Myoclonic dystonia 11 3 SGCE Autosomal recessive limb-girdle muscular dystrophy type 2C 1 SGCG Autosomal recessive limb-girdle muscular dystrophy type 2C 1 SGCG Walker-Warburg syndrome 13 SGK196 Sanfilippo syndrome type A 1 SGSH Distal 16pll.2 microdeletion syndrome 1 SH2B1 Proximal 16pll.2 microdeletion syndrome 1 SH2B1 Severe early-onset obesity-insulin resistance syndrome due t o SH2B1 deficiency 1 SH2B1 X-linked lymphoproliferative disease 2 SH2D1A Cherubism 1 SH3BP2 Frank-Ter Haar syndrome 1 SH3PXD2B Charcot-Marie-Tooth disease type 4 C 1 SH3TC2 Monosomy 22ql3 1 SHANK3 Split hand-split foot malformation 4 SH FM1 Alobar holoprosencephaly 14 SH H Colobomatous microphthalmia 8 SH H Hypoplastic tibiae - postaxial Polydactyly 2 SH H Lobar holoprosencephaly 14 SHH Microform holoprosencephaly 14 SHH Midline interhemispheric variant of holoprosencephaly 14 SH H Polydactyly of a triphalangeal thumb, bilateral 2 SH H Polydactyly of a triphalangeal thumb, unilateral · 2 SH H Radial hemimelia, bilateral 2 SHH Radial hemimelia, unilateral 2 SHH Schizencephaly 4 SH H Semilobar holoprosencephaly 14 SH H

161

Table IX-21781338.1 Septopreoptic holoprosencephaly 14 SH H Solitary median maxillary central incisor syndrome 1 SH H Syndactyly type 4 2 SH H Triphalangeal thumb - polysyndactyly syndrome 2 SH H Noonan-like syndrome with loose anagen hair 1 SHOC2 Langer mesomelic dysplasia 2 SHOX Leri-Weill dyschondrosteosis 1 SHOX Shox-related short stature 1 SHOX Intellectual deficit, X-linked, Stocco Dos Santos type 1 SHROOM4 Congenital sucrase-isomaltase deficiency with minimal starch tolerance 1 SI Congenital sucrase-isomaltase deficiency with starch and lactose intolerance 1 SI Congenital sucrase-isomaltase deficiency with starch intolerance 1 SI Congenital sucrase-isomaltase deficiency without starch intolerance 1 SI

Congenital sucrase-isomaltase deficiency without sucrose intolerance 1 SI Juvenile amyotrophic lateral sclerosis 3 SIGMAR1

Marinesco-Sjogren syndrome 1 SI LI

6ql6 deletion syndrome 1 SI M 1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 SIX1 BOR syndrome 3 SIX1 Branchio-otic syndrome 2 SIX1 Alobar holoprosencephaly 14 SIX3 Lobar holoprosencephaly 14 SIX3 Microform holoprosencephaly 14 SIX3 Midline interhemispheric variant of holoprosencephaly 14 SIX3 Schizencephaly 4 SIX3 Semilobar holoprosencephaly 14 SIX3 Septopreoptic holoprosencephaly 14 SIX3 BOR syndrome 3 SIX5 14q22q23 microdeletion syndrome 2 SIX6 Microphthalmia - cataract 4 SIX6 lp36 deletion syndrome 4 SKI Shprintzen-Goldberg syndrome 2 SKI Syndromic diarrhea 2 SKIV2L Tuberculosis 1 SLC11A1 Microcytic anemia with liver iron overload 1 SLC11A2 Antenatal Bartter syndrome 2 SLC12A1 Gitelman syndrome 2 SLC12A3 Corpus callosum agenesis - neuronopathy 1 SLC12A6 Exercise-induced hyperinsulinism 1 SLC16A1 Exercise-induced hyperinsulinism 1 SLC16A1

162

Table IX-21781338.1 Metabolic myopathy due to lactate transporter defect 1 SLC16A1 Juvenile cataract - microcornea - renal glucosuria 1 SLC16A12 Allan-Hemdon-Dudley syndrome 1 SLC16A2 Free sialic acid storage disease, infantile form 1 SLC17A5 Intermediate severe Salla disease 1 SLC17A5 Salla disease 1 SLC17A5 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 SLC17A8 Thiamine-responsive megaloblastic anemia syndrome 1 SLC19A2 Biotin-responsive basal ganglia disease 1 SLC19A3 Infantile spams - psychomotor retardation - progressive brain atrophy - basal ganglia disease 1 SLC19A3 Leigh syndrome with leukodystrophy 13 SLC19A3 Thiamine-responsive encephalopathy 1 SLC19A3 Alternating hemiplegia of childhood 4 SLC1A3 Episodic ataxia type 6 1 SLC1A3 Bilateral striopallidodentate calcinosis 2 SLC20A2 Hereditary renal hypouricemia 2 SLC22A12 Carnitine uptake deficiency 1 SLC22A5 Congenital stationary night blindness 12 SLC24A1 Hypocalcified amelogenesis imperfecta 2 SLC24A4 D,L-2-hydroxyglutaric aciduria 1 SLC25A1 Epileptic encephalopathy with global cerebral demyelination 1 SLC25A12 Citrullinemia type II 1 SLC25A13 Neonatal intrahepatic cholestasis due to citrin deficiency 1 SLC25A13 Hyperornithinemia-hyperammonemia-homocitrullinuria 1 SLC25A15 Amish lethal microcephaly 1 SLC25A19 Progressive demyelinating neuropathy with bilateral striatal necrosis 1 SLC25A19 Carnitine-acylcarnitine translocase deficiency 1 SLC25A20 Early infantile epileptic encephalopathy 9 SLC25A22 Early myoclonic encephalopathy 1 SLC25A22 Cardiomyopathy - hypotonia - lactic acidosis 1 SLC25A3 Autosomal recessive pyridoxine-refractory sideroblastic anemia 2 SLC25A38 Autosomal dominant progressive external ophthalmoplegia 5 SLC25A4 Congenital cataract - hypertrophic cardiomyopathy - mitochondrial myopathy 2 SLC25A4 Facioscapulohumeral dystrophy 4 SLC25A4 Achondrogenesis type I B 1 SLC26A2 Atelosteogenesis type II 1 SLC26A2 Diastrophic dwarfism 1 SLC26A2 Multiple epiphyseal dysplasia type 4 1 SLC26A2 Congenital chloride diarrhea 1 SLC26A3

163

Table IX-21781338.1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SLC26A4 Pendred syndrome 3 SLC26A4 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SLC26A5 Ichthyosis prematurity syndrome 1 SLC27A4 Dysosteosclerosis 1 SLC29A3 Faisalabad histiocytosis 1 SLC29A3 Familial sinus histiocytosis with massive lymphadenopathy 1 SLC29A3 H syndrome 1 SLC29A3 Pigmented hypertrichosis with insulin dependent diabetes mellitus syndrome 1 SLC29A3 Childhood absence epilepsy 6 SLC2A1 Encephalopathy due to GLUT1 deficiency 1 SLC2A1 Epilepsy with myoclonic-astatic seizures 2 SLC2A1 Hereditary cryohydrocytosis with reduced stomatin 1 SLC2A1 Paroxysmal dystonic choreathetosis with episodic ataxia and spasticity 1 SLC2A1 Paroxysmal exertion-induced dyskinesia 1 SLC2A1 Arterial tortuosity syndrome 1 SLC2A10 Glycogen storage disease due to GLUT2 deficiency 1 SLC2A2 Hereditary renal hypouricemia 2 SLC2A9 Cirrhosis-dystonia-polycythemia-hypermanganesemia syndrome 1 SLC30A10 . Autosomal dominant spastic paraplegia type 42 1 SLC33A1 Congenital cataract-hearing loss-severe developmental delay syndrome 1 SLC33A1 Dominant hypophosphatemia with nephrolithiasis or osteoporosis 2 SLC34A1 Primary Fanconi syndrome 1 SLC34A1 Pulmonary alveolar microlithiasis 1 SLC34A2 Pulmonary alveolar microlithiasis 1 SLC34A2 Hereditary hypophosphatemic rickets with hypercalciuria 1 SLC34A3 SLC35A1-CDG syndrome 1 SLC35A1 SLC35A2-CDG syndrome 1 SLC35A2 Leukocyte adhesion deficiency type II 1 SLC35C1 Schneckenbecken dysplasia 1 SLC35D1 Iminoglycinuria 4 SLC36A2 Glycogen storage disease due to glucose-6-phosphatase deficiency type b 1 SLC37A4 Ehlers-Danlos syndrome, spondylocheiro dysplastic type 1 SLC39A13 Acrodermatitis enteropathica, zinc deficiency type 1 SLC39A4 2p21 microdeletion syndrome 4 SLC3A1 Atypical hypotonia - cystinuria syndrome 3 SLC3A1 Cystinuria type A 1 SLC3A1 Hypotonia - cystinuria syndrome 2 SLC3A1 Hemochromatosis type 4 1 SLC40A1 Oculocutaneous albinism type 4 1 SLC45A2

Table IX-21781338.1 Hereditary folate malabsorption 1 SLC46A1 Autosomal dominant distal renal tubular acidosis 1 SLC4A1 Distal renal tubular acidosis with anemia 1 SLC4A1 Hereditary elliptocytosis 3 SLC4A1 Hereditary spherocytosis 5 SLC4A1

Congenital hereditary endothelial dystrophy II 1 SLC4A11 Corneal dystrophy - perceptive deafness 1 SLC4A11 Fuchs endothelial corneal dystrophy 4 SLC4A11 Transient neonatal multiple acyl-CoA dehydrogenase deficiency 1 SLC52A1 Riboflavin transporter deficiency 2 SLC52A2 Riboflavin transporter deficiency 2 SLC52A3 Glucose-galactose malabsorption 1 SLC5A1 Renal glucosuria 1 SLC5A2 Familial thyroid dyshormonogenesis 6 SLC5A5 Distal hereditary motor neuropathy type 7 2 SLC5A7 Iminoglycinuria 4 SLC6A18 Hartnup syndrome 1 SLC6A19 Iminoglycinuria 4 SLC6A19 Iminoglycinuria 4 SLC6A20 Infantile dystonia-parkinsonism 1 SLC6A3 Hereditary hyperekplexia 4 SLC6A5 X-linked creatine transporter deficiency 1 SLC6A8 Lysinuric protein intolerance 1 SLC7A7 Cystinuria type B 1 SLC7A9 Dominant hypophosphatemia with nephrolithiasis o r osteoporosis 2 SLC9A3R1 Christianson syndrome 1 SLC9A6 Rotor syndrome 2 SLC01B1 Rotor syndrome 2 SLC01B3 Pachydermoperiostosis 2 SLC02A1 Mesomelia-synostoses syndrome 2 SLC05A1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SLITRK6 ai de Meleda 1 SLURP1 Fanconi anemia 16 SLX4 Aneurysm - osteoarthritis syndrome 1 SMAD3 Familial thoracic aortic aneurysm and aortic dissection 8 SMAD3 Familial pancreatic carcinoma 8 SMAD4 Generalized juvenile polyposis/juvenile polyposis coli 3 SMAD4 Myhre syndrome 1 SMAD4 Rendu-Osler-Weber disease 3 SMAD4 Heritable pulmonary arterial hypertension 5 SMAD9 Intellectual deficit - sparse hair - brachydactyly 1 SMARCA2

165

Table IX-21781338.1 Coffin-Siris syndrome 4 SMARCA4 Familial rhabdoid tumor 2 SMARCA4 Isolated adermatoglyphia 1 SMARCAD1 Schimke immuno-osseous dysplasia 1 SMARCAL1 Atypical teratoid tumor 1 SMARCB1 Coffin-Siris syndrome 4 SMARCB1 Familial multiple meningioma 5 SMARCB1 Familial rhabdoid tumor 2 SMARCB1 Neurofibromatosis type 3 2 SMARCB1 Familial multiple meningioma 5 SMARCE1 Cornelia de Lange syndrome 5 SMC1A Cornelia de Lange syndrome 5 SMC3 Facioscapulohumeral dystrophy 4 SMCHD1 Proximal spinal muscular atrophy type 1 3 SMN 1 Proximal spinal muscular atrophy type 2 3 SMN 1 Proximal spinal muscular atrophy type 3 3 SMN1 Proximal spinal muscular atrophy type 4 2 SMN 1 Proximal spinal muscular atrophy type 1 3 SMN2 Proximal spinal muscular atrophy type 2 3 SMN2 Proximal spinal muscular atrophy type 3 3 SMN2 Proximal spinal muscular atrophy type 4 2 SMN2 Microphthalmia with limb anomalies 1 SMOC1 Atypical dentin dysplasia due to SMOC2 deficiency 1 SMOC2 Niemann-Pick disease type A 1 SMPD1 Niemann-Pick disease type B 1 SMPD1 X-linked nonsyndromic sensorineural deafness type DFN 2 SMPX Intellectual deficit, X-linked, Snyder type 1 SMS Piebaldism 2 SNAI2 Waardenburg syndrome type 2 4 SNAI2 CEDNI K syndrome 1 SNAP29 Parkinsonian-pyramidal syndrome 2 SNCA Young adult-onset Parkinsonism 13 SNCA SNORD11S Prader-Willi syndrome 6 @ SNORD116 Prader-Willi syndrome 6 @ Retinitis pigmentosa 6 1 SNRNP200 Hypotrichosis simplex 6 SNRPE Angelman syndrome 6 SNRPN Prader-Willi syndrome 6 SNRPN Romano-Ward syndrome 13 SNTA1

166

Table IX-21781338.1 Autosomal recessive malignant osteopetrosis 5 SNX10 MMEP syndrome 1 SNX3 Amyotrophic lateral sclerosis 23 SOD1 Male infertility with normal virilization due to meiosis defect 2 SOHLH1 Early-onset autosomal dominant Alzheimer disease 4 SORL1 Hereditary gingival fibromatosis 1 SOS1 Noonan syndrome 10 SOS1 Craniodiaphyseal dysplasia 1 SOST Hyperostosis corticalis generalisata 2 SOST Sclerosteosis 2 SOST Kallmann syndrome 19 SOX10 Neurologic Waardenburg-Shah syndrome 1. SOX10 Waardenburg syndrome type 2 4 SOX10 Waardenburg-Shah syndrome 3 SOX10 Familial vesicoureteral reflux 2 SOX17 Hypotrichosis - lymphedema - telangiectasia 1 . SOX18 Anophthalmia/microphthalmia - esophageal atresia 1 SOX2 Colobomatous microphthalmia 8 SOX2 Septo-optic dysplasia 6 SOX2 Panhypopituitarism 2 SOX3 Septo-optic dysplasia 6 SOX3 X-linked congenital generalized hypertrichosis 1 SOX3 X-linked intellectual deficit with isolated growth hormone deficiency 1 SOX3 12pl2.1 microdeletion syndrome 1 SOX5 Developmental and speech delay due to SOX5 deficiency 1 SOX5 46,XX ovotesticular disorder of sex development 1 SOX9 Campomelic dysplasia 1 SOX9 Hepatic veno-occlusive disease - immunodeficiency 1 SP110 Osteogenesis imperfecta type 4 7 SP7 Autosomal dominant spastic paraplegia type 4 1 SPAST Globozoospermia 2 SPATA16 Leber congenital amaurosis 18 SPATA7 Retinitis pigmentosa 6 1 SPATA7 Commissural facial cleft 2 SPECC1L Tessier number 4 facial cleft 1 SPECC1L Autosomal recessive spastic paraplegia type 11 1 SPG 11 Autosomal recessive spastic paraplegia type 14 1 SPG14 X-linked spastic paraplegia type 16 1 SPG16 Autosomal dominant spastic paraplegia type 19 1 SPG19 Autosomal recessive spastic paraplegia type 20 1 SPG20 Autosomal recessive spastic paraplegia type 21 1 SPG21

167

Table IX-21781338.1 Autosomal recessive spastic paraplegia type 21 1 SPG21 Autosomal recessive spastic paraplegia type 23 1 SPG23 Autosomal recessive spastic paraplegia type 23 1 SPG23 Autosomal recessive spastic paraplegia type 24 1 SPG 24 Autosomal recessive spastic paraplegia type 24 1 SPG24 Autosomal recessive spastic paraplegia type 25 1 SPG 25 Autosomal recessive spastic paraplegia type 27 1 SPG27 Autosomal dominant spastic paraplegia type 29 1 SPG29 Autosomal recessive spastic paraplegia type 32 1 SPG32 X-linked spastic paraplegia type 34 1 SPG34 Autosomal dominant spastic paraplegia type 36 1 SPG36 Autosomal dominant spastic paraplegia type 37 1 SPG37 Autosomal dominant spastic paraplegia type 38 1 SPG38 Autosomal dominant spastic paraplegia type 4 1 1 SPG41 Autosomal recessive spastic paraplegia type 43 1 SPG43 Autosomal recessive spastic paraplegia type 45 1 SPG45 Autosomal dominant optic atrophy and peripheral neuropathy 1 SPG7 Autosomal recessive spastic paraplegia type 7 1 SPG7 Autosomal dominant spastic paraplegia type 9 1 SPG9 Primary biliary cirrhosis 8 SPIB Hereditary chronic pancreatitis 6 SPI NK1 Netherton syndrome 1 SPI NK5 Congenital sodium diarrhea 1 SPINT2 Dopa responsive dystonia due t o sepiapterin reductase deficiency 1 SPR Legius syndrome 1 SPRED1 Kallmann syndrome 19 SPRY4 Normosmic congenital hypogonadotropic hypogonadism 18 SPRY4 Common hereditary elliptocytosis 1 SPTA1 Hereditary elliptocytosis 3 SPTA1 Hereditary pyropoikilocytosis 1 SPTA1 Hereditary spherocytosis 5 SPTA1 Early infantile epileptic encephalopathy 9 SPTAN1 Hereditary spherocytosis 5 SPTB Spectrin-associated autosomal recessive cerebellar ataxia 1 SPTBN2 Spinocerebellar ataxia type 5 1 SPTBN2 Hereditary sensory and autonomic neuropathy type 1 4 SPTLC1 Hereditary sensory and autonomic neuropathy type 1 4 SPTLC2 Floating-Harbor syndrome 1 SRCAP 46,XY disorder of sex development due to 5-alpha-reductase 2 deficiency 1 SRD5A2 Familial prostate cancer 14 SRD5A2 5RD5A3-CDG syndrome 1 SRD5A3

Table IX-21781338.1 Autosomal dominant aplasia and myelodysplasia 1 SRP72 Benign familial epilepsy of childhood with rolandic spikes 1 SRPX2 Bilateral perisylvian polymicrogyria 1 SRPX2 Rolandic epilepsy - speech dyspraxia 1 SRPX2 45,X/46,XY mixed gonadal dysgenesis 2 SRY 46,XX testicular disorder of sex development 1 SRY 46,XY complete gonadal dysgenesis 8 SRY 46,XY partial gonadal dysgenesis 7 SRY Testicular regression syndrome 1 SRY Synovial sarcoma 3 SS18 Acromegaly 2 SSTR5 Synovial sarcoma 3 SSX1 Synovial sarcoma 3 SSX2 Ichthyosis-hypotrichosis syndrome 1 ST14 Autosomal recessive nonsyndromic intellectual deficit 15 ST3GAL3 West syndrome 4 ST3GAL3 Amish infantile epilepsy syndrome 1 ST3GAL5 Native American myopathy 1 STAC3 Microcephaly-capillary malformation syndrome 1 STAMBP Classic congenital lipoid adrenal hyperplasia due t o STAR deficency 1 STAR Familial glucocorticoid deficiency 4 STAR Non-classic congenital lipoid adrenal hyperplasia due to STAR deficency 1 STAR Chronic mucocutaneous candidiasis 6 STATl Mendelian susceptibility to mycobacterial diseases due t o partial STATl deficiency 1 STATl Autosomal dominant hyper IgE syndrome 1 STAT3 Behcet disease 9 STAT4 Juvenile rheumatoid factor-negative polyarthritis 9 STAT4 Oligoarticular juvenile arthritis 9 STAT4 Laron syndrome with immunodeficiency 1 STAT5B Solitary fibrous tumor 2 STAT6 Severe congenital hypochromic anemia with ringed sideroblasts 1 STEAP3 Autosomal recessive primary microcephaly 11 STIL Precursor T-cell acute lymphoblastic leukemia 19 STIL Combined immunodeficiency due t o STIM1 deficiency 1 STIM 1 Combined immunodeficiency due t o STIM1 deficiency 1 STIM 1 Tubular aggregate myopathy 1 STIM 1 Peutz-Jeghers syndrome 1 STK11 Combined immunodeficiency due t o STK4 deficiency 1 STK4 Colobomatous microphthalmia 8 STRA6 Matthew-Wood syndrome 1 STRA6

Table IX-21781338.1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 ST C Deafness-infertility syndrome 2 STRC Recessive X-linked ichthyosis 1 STS Syndromic X-linked ichthyosis 1 STS Familial hemophagocytic lymphohistiocytosis 4 STX11 Pseudohypoparathyroidism type B 2 STX16 Early infantile epileptic encephalopathy 9 STXBP1 Familial hemophagocytic lymphohistiocytosis 4 STXBP2 Mitochondrial DNA depletion syndrome, encephalomyopathic form with methylmalonic aciduria 1 SUCLA2 Fatal infantile lactic acidosis with methylmalonic aciduria 1 SUCLG1 Desmoplastic/nodular medulloblastoma 1 SUFU Familial multiple meningioma 5 SUFU Medulloblastoma with extensive nodularity 1 SUFU Mesomelia-synostoses syndrome 2 SULF1 Multiple sulfatase deficiency 1 SUMF1 Isolated sulfite oxidase deficiency 1 SUOX Fatal infantile cytochrome C oxidase deficiency 5 SURF1 Leigh syndrome with cardiomyopathy 8 SURF1 Leigh syndrome with leukodystrophy 13 SURF1 17qll microdeletion syndrome 3 SUZ12 Male infertility with normal virilization due to meiosis defect 2 SYCP3 X-linked epilepsy - learning disabilities - behavior disorders 1 SYN1 Autosomal dominant Emery-Dreifuss muscular dystrophy 4 SYNE 1 Autosomal recessive ataxia, Beauce type 1 SYNE1 Autosomal recessive myogenic arthrogryposis multiplex congenita 1 SYNE1 Autosomal dominant Emery-Dreifuss muscular dystrophy 4 SYNE2 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SYNE4 Autosomal dominant nonsyndromic intellectual deficit 15 SYNGAP1 X-linked nonsyndromic intellectual deficit 24 SYP Autosomal recessive cerebellar ataxia - psychomotor retardation 1 SYT14 Cervical spina bifida aperta 6 T Cervical spina bifida cystica 6 T Cervicothoracic spina bifida aperta 6 T Cervicothoracic spina bifida cystica 6 T Chordoma 1 T Lumbosacral spina bifida aperta 6 T Lumbosacral spina bifida cystica 6 T Thoracolumbosacral spina bifida aperta 6 T Thoracolumbosacral spina bifida cystica 6 T Total spina bifida aperta 6 T

Table IX-21781338.1 Total spina bifida cystica 6 T Upper thoracic spina bifida aperta 6 T Upper thoracic spina bifida cystica 6 T Normosmic congenital hypogonadotropic hypogonadism 18 TAC3 Giant cell glioblastoma 10 TACC1 Gliosarcoma 10 TACC1 Giant cell glioblastoma 10 TACC3 Gliosarcoma 10 TACC3 Leigh syndrome with cardiomyopathy 8 TACOl Kallmann syndrome 19 TAC 3 Normosmic congenital hypogonadotropic hypogonadism 18 TACR3 Gelatinous drop-like corneal dystrophy 1 TACSTD2 X-linked dystonia-parkinsonism 1 TAF1 Precursor T-cell acute lymphoblastic leukemia 19 TALI Transaldolase deficiency 1 TALDOl Immunodeficiency by defective expression of HLA class 1 3 TAP1 Immunodeficiency by defective expression of HLA class 1 3 TAP2 Immunodeficiency by defective expression of HLA class 2 5 TAP2 Immunodeficiency by defective expression of HLA class 1 3 TAPBP Amyotrophic lateral sclerosis 23 TARDBP Frontotemporal dementia with motor neuron disease 3 TARDBP Tyrosinemia type 2 1 TAT Barth syndrome 1 TAZ Familial isolated dilated cardiomyopathy 38 TAZ Left ventricular noncompaction 11 TAZ Familial infantile myoclonic epilepsy 1 TBC1D24 Focal epilepsy - intellectual deficit - cerebro-cerebellar malformation 1 TBC1D24 Malignant migrating partial seizures of infancy 4 TBC1D24 Progressive myoclonic epilepsy with dystonia 1 TBC1D24 Autosomal recessive Kenny-Caffey syndrome 1 TBCE Sanjad-Sakati syndrome 1 TBCE Herpetic encephalitis 5 TBK1 Williams syndrome 17 TBL2 Spinocerebellar ataxia type 17 1 TBP 22qll.2 deletion syndrome 7 TBX1 22qll.2 microduplication syndrome 1 TBX1 Pelviscapular dysplasia 1 TBX15 Congenital isolated ACTH deficiency 1 TBX19 17q23.1q23.2 microdeletion syndrome 2 TBX2 Atrial septal defect, ostium secundum type 8 TBX20 Abruzzo-Erickson syndrome 1 TBX22

171

Table IX-21781338.1 X-linked cleft palate and ankyloglossia 1 TBX22 Ulnar-mammary syndrome 1 TBX3 17q23.1q23.2 microdeletion syndrome 2 TBX4 Coxo-podo-patellar syndrome 1 TBX4 Familial clubfoot due to 17q23.1q23.2 microduplication 1 TBX4 Familial clubfoot due to 17q23.1q23.2 microduplication 1 TBX4 Holt-Oram syndrome 1 TBX5 Autosomal dominant spondylocostal dysostosis 1 TBX6 Bleeding diathesis due to thromboxane synthesis deficiency 1 TBXA2 Ghosal hematodiaphyseal dysplasia 1 TBXAS1 Autosomal recessive limb-girdle muscular dystrophy type 2G 1 TCAP Familial isolated dilated cardiomyopathy 38 TCAP Isolated brachycephaly 3 TCF12 Isolated plagiocephaly 3 TCF12 Saethre-Chotzen syndrome 4 TCF12 Precursor B-cell acute lymphoblastic leukemia 13 TCF3 Autosomal dominant nonsyndromic intellectual deficit 15 TCF4 Fuchs endothelial corneal dystrophy 4 TCF4 Pallister-Hall syndrome 2 TCF4 Pitt-Hopkins syndrome 1 TCF4 Autosomal recessive malignant osteopetrosis 5 TCI RG1 Precursor T-cell acute lymphoblastic leukemia 19 TCL1A Transcobalamin II deficiency 1 TCN2 Treacher-Collins syndrome 3 TCOF1 Joubert syndrome 9 TCTN1 Joubert syndrome with renal defect 5 TCTN2 Meckel syndrome 13 TCTN2 Joubert syndrome with orofaciodigital defect 4 TCTN3 Oral-facial-digital syndrome type 4 1 TCTN3 Alobar holoprosencephaly 14 TDGF1 Lobar holoprosencephaly 14 TDGF1 Microform holoprosencephaly 14 TDGF1 Midline interhemispheric variant of holoprosencephaly 14 TDGF1 Semilobar holoprosencephaly 14 TDGF1 Septopreoptic holoprosencephaly 14 TDGF1 Spinocerebellar ataxia type 1 with axonal neuropathy 1 TDP1 Helicoid peripapillary chorioretinal degeneration 1 TEADl Autosomal recessive spastic paraplegia type 49 1 TECPR2 Autosomal recessive nonsyndromic intellectual deficit 15 TECR Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 TECTA Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TECTA

172

Table IX-21 78 338.1 Mucocutaneous venous malformations 1 TEK Dyskeratosis congenita 10 TERC Idiopathic aplastic anemia 5 TERC Idiopathic pulmonary fibrosis 11 TERC Dyskeratosis congenita 10 TERT Familial melanoma 6 TERT Hoyeraal-Hreidarsson syndrome 4 TERT Idiopathic aplastic anemia 5 TERT Idiopathic pulmonary fibrosis 11 TERT Acquired idiopathic sideroblastic anemia 1 TET2 Acute myeloid leukemia with multilineage dysplasia 1 TET2 Essential thrombocythemia 4 TET2 Myelofibrosis with myeloid metaplasia 3 TET2 Polycythemia vera 3 TET2 Refractory anemia 1 TET2 Refractory anemia with excess blasts 1 TET2 Congenital atransferrinemia 1 T F Branchio-oculo-facial syndrome 1 TFAP2A Char syndrome 1 TFAP2B Mitochondrial nonsyndromic sensorineural deafness 6 TFB1M Alveolar soft-part sarcoma 2 TFE3 Translocation renal cell carcinoma 7 TFE3 Translocation renal cell carcinoma 7 TFEB Hereditary motor and sensory neuropathy, Okinawa type 1 TFG Papillary or follicular thyroid carcinoma 14 TFG Spastic paraplegia-optic atrophy-neuropathy syndrome 1 TFG Hemochromatosis type 3 1 TFR2 Familial thyroid dyshormonogenesis 6 TG Oligodontia 10 TGFA Camurati-Engelmann disease 1 TGFB1 Cystic fibrosis 4 TGFB1 Familial thoracic aortic aneurysm and aortic dissection 8 TGFB2 Peters anomaly 6 TGFB2 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 TGFB3 Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 TGFB3 Familial isolated arrhythmogenic ventricular dysplasia, right dominant form I P TGFB3 Granular corneal dystrophy type 1 1 TGFBI

Granular corneal dystrophy type II 1 TGFBI Microcystic corneal dystrophy 1 TGFBI Reis-Bucklers corneal dystrophy 1 TGFBI

173

Table IX-21781338.1 Familial thoracic aortic aneurysm and aortic dissection 8 TGFBR1 Loeys-Dietz syndrome type 1 2 TGFBR1 Multiple keratoacanthoma, Ferguson-Smith type 1 TGFBR1 Aortic dilatation - joint hypermobility - arterial tortuosity 1 TGFBR2 Familial thoracic aortic aneurysm and aortic dissection 8 TGFBR2 Hereditary nonpolyposis colon cancer 11 TGFBR2 Loeys-Dietz syndrome type 1 2 TGFBR2 Marfan syndrome type 2 1 TGFBR2 Alobar holoprosencephaly 14 TGIF1 Lobar holoprosencephaly 14 TGIF1 Microform holoprosencephaly 14 TGIF1 Midline interhemispheric variant of holoprosencephaly 14 TGIF1 Semilobar holoprosencephaly 14 TGIF1 Septopreoptic holoprosencephaly 14 TGIF1 Acral self-healing collodion baby 1 TGM1 Bathing suit ichthyosis 1 TGM1 Congenital nonbullous ichthyosiform erythroderma 6 TGM1 Lamellar ichthyosis 6 TGM1 Self-healing collodion baby 3 TGM1 Localized peeling skin syndrome 1 TGM5 Spinocerebellar ataxia type 35 1 TGM6 Autosomal recessive dopa-responsive dystonia 1 TH Primary dystonia, DYT6 type 1 THAP1 Atypical hemolytic uremic syndrome with thrombomodulin anomaly 1 THBD Familial thrombomodulin anomalies 1 THBD Familial thrombocytosis 3 THPO Hereditary thrombocytosis with transverse limb defect 1 THPO Peripheral resistance t o thyroid hormones 2 THRA Generalized resistance to thyroid hormone 1 THRB Peripheral resistance t o thyroid hormones 2 THRB Selective pituitary resistance to thyroid hormone 1 THRB Distal myopathy, Welander type 1 TIA1 Distal myopathy, Welander type 1 TIA1 Herpetic encephalitis 5 TICAM1 Mohr-Tranebjaerg syndrome 1 TIMM8A Sorsby's fundus dystrophy 1 TIM P3 Dyskeratosis congenita 10 TINF2 Hoyeraal-Hreidarsson syndrome 4 TINF2 Retinopathy - anemia- central nervous system anomalies 1 TIN F2 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 TJP2 Familial hypercholanemia 3 TJP2

174

Table IX-21781338.1 Autosomal recessive progressive external ophthalmoplegia 2 TK2 Mitochondrial DNA depletion syndrome, myopathic form 1 TK2 Atrial septal defect, ostium primum type 1 TLL1 Atrial septal defect, ostium secundum type 8 TLL1 Herpetic encephalitis 5 TLR3 Behcet disease 9 TLR4 Precursor T-cell acute lymphoblastic leukemia 19 TLX1 Precursor T-cell acute lymphoblastic leukemia 19 TLX3 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 TMC1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TMC1 Epidermodysplasia verruciformis 2 TMC6 Epidermodysplasia verruciformis 2 TMC8 TMCOl defect syndrome 1 TMCOl Microphthalmia - cataract 4 TMEM 114 Autosomal recessive optic atrophy, OPA7 type 1 TMEM 126A Hereditary pheochromocytoma-paraganglioma 7 TMEM127 Joubert syndrome with oculorenal defect 6 TMEM138 TMEM 165-CDG syndrome 1 TMEM165 FRAXF syndrome 1 TMEM185A Joubert syndrome with oculorenal defect 6 TMEM216 Jou bert syndrome with orofaciodigital defect 4 TMEM216 Meckel syndrome 13 TMEM216 Joubert syndrome 9 TMEM231 Joubert syndrome with ocular defect 5 TMEM231 Meckel syndrome 13 TMEM231 Joubert syndrome 9 TMEM237 Joubert syndrome with ocular defect 5 TMEM237 Joubert syndrome with oculorenal defect 6 TMEM237 Joubert syndrome with renal defect 5 TMEM237 Osteogenesis imperfecta type 4 7 TMEM38B Autosomal dominant Emery-Dreifuss muscular dystrophy 4 TMEM43 Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 TMEM43 Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 TMEM43 Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 TMEM43 Walker-Warburg syndrome 13 TMEM5 Joubert syndrome 9 TMEM67 Joubert syndrome with hepatic defect 3 TMEM67 Meckel syndrome 13 TMEM67 ' Senior-Boichis syndrome 1 TMEM67 Isolated ATP synthase deficiency 4 TMEM70

175

Table IX-21781338.1 Mitochondrial encephalo-cardio-myopathy due to TMEM70 deficiency 1 TMEM70 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TMIE Familial isolated dilated cardiomyopathy 38 TMPO Congenital enteropathy due to enteropeptidase deficiency 1 TMPRSS15 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TMPRSS3

IRI DA syndrome 1 TMPRSS6 Squamous cell carcinoma of head and neck 4 TNFRSF10B Autosomal recessive malignant osteopetrosis 5 TNFRSF11A Familial expansile osteolysis 1 TNFRSF11A Osteopetrosis - hypogammaglobulinemia 1 TNFRSF11A Juvenile Paget's disease 1 TNFRSF11B Common variable immunodeficiency 10 TNFRSF13B Common variable immunodeficiency 10 TNFRSF13C Intermittent hydrarthrosis 2 TNFRSF1A TRAPS syndrome 1 TNFRSF1A Autosomal recessive malignant osteopetrosis 5 TNFSF11 Common variable immunodeficiency 10 TNFSF12 Primary biliary cirrhosis 8 TNFSF15 Familial isolated dilated cardiomyopathy 38 TNNC1 Digitotalar dysmorphism 5 TNNI2 Sheldon-Hall syndrome 4 TNNI2 Familial isolated dilated cardiomyopathy 38 TNNI3 Familial isolated restrictive cardiomyopathy 3 TNNI3 Amish nemaline myopathy 1 TNNT1 Familial isolated dilated cardiomyopathy 38 TNNT2 Familial isolated restrictive cardiomyopathy 3 TNNT2 Left ventricular noncompaction 11 TN T2 Digitotalar dysmorphism 5 TNNT3 Sheldon-Hall syndrome 4 TNNT3 Autosomal dominant limb-girdle muscular dystrophy type I F 1 TNP03 Primary biliary cirrhosis 8 TNP03 Ehlers-Danlos syndrome due to tenascin-X deficiency 1 TNXB Ehlers-Danlos syndrome, hypermobility type 1 TNXB Neuroblastoma 7 TOP2A Retinitis pigmentosa 6 1 TOPORS Early onset torsion dystonia 1 TOR1A Myoclonic dystonia 11 3 TOR1A Adrenocortical carcinoma 1 TP53 B-cell chronic lymphocytic leukemia 7 TP53 Essential thrombocythemia 4 TP53 Familial pancreatic carcinoma 8 TP53

176

Table IX-21781338.1 Giant cell glioblastoma 10 TP53 Gliosarcoma 10 TP53 Li-Fraumeni syndrome 2 TP53 Papilloma of choroid plexus 1 TP53 ADULT syndrome 1 TP63 Ankyloblepharon - ectodermal defects - cleft lip/palate 1 TP63 Bladder exstrophy 1 TP63 EEC syndrome 1 TP63 Limb-mammary syndrome 1 TP63 Split hand-split foot malformation 4 TP63 Triose phosphate-isomerase deficiency 1 TPI 1 Childhood encephalopathy due t o thiamine pyrophosphokinase deficiency 1 TPK1 Familial isolated dilated cardiomyopathy 38 TPM1 Left ventricular noncompaction 11 TPM1 Cap myopathy 2 TPM2 Childhood-onset nemaline myopathy 5 TP Congenital fiber-type disproportion myopathy 5 TPM2 Digitotalar dysmorphism 5 TPM2 Sheldon-Hall syndrome 4 TPM2 Trismus - pseudocamptodactyly 2 TPM2 Typical nemaline myopathy 4 TPM2 Childhood-onset nemaline myopathy 5 TPM3 Congenital fiber-type disproportion myopathy 5 TPM3 Inflammatory myofibroblastic tumor 6 TPM3 Intermediate nemaline myopathy 3 TPM3 Inflammatory myofibroblastic tumor 6 TPM4 Thiopurine S-methyltransferase deficiency 1 TP T Familial thyroid dyshormonogenesis 6 TPO Childhood-onset autosomal recessive slowly progressive spinocerebellar ataxia 1 TPP1 CLN2 disease 1 TPP1 CLN2 disease 1 TPP1 Papillary or follicular thyroid carcinoma 14 TPR Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TPRN Precursor T-cell acute lymphoblastic leukemia 19 TRA Herpetic encephalitis 5 TRAF3 Autosomal dominant hypohidrotic ectodermal dysplasia 3 TRAF6 Spondyloepiphyseal dysplasia tarda 1 TRAPPC2 Autosomal recessive nonsyndromic intellectual deficit 15 TRAPPC9 Intellectual deficit - obesity - brain malformations - facial dysmorphism 1 TRAPPC9 Precursor T-cell acute lymphoblastic leukemia 19 TRB

177

Table IX-21781338.1 Precursor T-cell acute lymphoblastic leukemia 19 TRD Catecholaminergic polymorphic ventricular tachycardia 4 TRDN Diarrhea-vomiting due t o trehalase deficiency 1 TREH Nasu-Hakola disease 2 TREM2 Aicardi-Goutieres syndrome 6 TREX1 Cerebroretinal vasculopathy 1 TREX1 Chil blain lupus 2 TREX1 Hereditary vascular retinopathy 1 TREX1 HERNS syndrome 1 TREX1 Precursor T-cell acute lymphoblastic leukemia 19 TRG Precursor T-cell acute lymphoblastic leukemia 19 TRG Isolated thyrotropin-releasing hormone deficiency 1 TRH Resistance to thyrotropin-releasing hormone syndrome 1 TRHR Papillary or follicular thyroid carcinoma 14 TRIM24 Papillary or follicular thyroid carcinoma 14 TRIM27 Autosomal recessive limb-girdle muscular dystrophy type 2H 1 TRIM32 Bardet-Biedl syndrome 17 TRIM32 Papillary or follicular thyroid carcinoma 14 TRIM33 MULIBREY nanism 1 TRIM37 Autosomal recessive nonsyndromic sensorineural deafness type DFN B 52 TRIOBP Achondrogenesis type 1A 1 TRIP11 Acute infantile liver failure due to mtDNA-encoded proteins synthesis defect 1 TRMU Mitochondrial myopathy with reversible cytochrome C oxidase deficiency 2 TRMU Mitochondrial nonsyndromic sensorineural deafness 6 TRMU Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 TRPC6 Congenital stationary night blindness 12 TRPM1 Brugada syndrome 12 TRPM4 Familial progressive cardiac conduction defect 3 TRPM4 Autosomal recessive primary hypomagnesemia with normocalcuria and hypocalcemia 1 TRPM6 Amyotrophic lateral sclerosis-parkinsonism-dementia complex 2 TRPM7 Langer-Giedion syndrome 2 TRPS1 Trichorhinophalangeal syndrome type 1 and 3 1 TRPS1 Mutilating palmoplantar keratoderma with periorificial keratotic plaques 2 TRPV3 Autosomal dominant Charcot-Marie-Tooth disease type 2C 1 TRPV4 Autosomal dominant congenital benign spinal muscular atrophy 1 TRPV4 Brachyolmia type 3 1 TRPV4 Familial digital arthropathy-brachydactyly 1 TRPV4 Metatropic dysplasia type 1 1 TRPV4 Parastremmatic dwarfism 1 TRPV4 178

Table IX-21781338.1 Scapuloperoneal amyotrophy ' 4 TRPV4 Spondyloepiphyseal dysplasia, Maroteaux type 1 TRPV4 Spondylometaphyseal dysplasia, Kozlowski type 1 TRPV4 Isolated focal cortical dysplasia 1 TSC1 Isolated focal cortical dysplasia 1 TSC1 Lymphangioleiomyomatosis 2 TSC1 Tuberous sclerosis 2 TSC1 Autosomal dominant polycystic kidney disease type 1 with tuberous sclerosis 2 TSC2 Lymphangioleiomyomatosis 2 TSC2 Tuberous sclerosis 2 TSC2 Pontocerebellar hypoplasia type 2 4 TSEN2 Pontocerebellar hypoplasia type 2 4 TSEN34 Pontocerebellar hypoplasia type 1 4 TSEN54 Pontocerebellar hypoplasia type 2 4 TSEN54 Pontocerebellar hypoplasia type 4 1 TSEN54 Pontocerebellar hypoplasia type 5 1 TSEN54 Fatal mitochondrial disease due to combined oxidative phosphorylation deficiency 3 1 TSFM Isolated thyroid-stimulating hormone deficiency 1 TSHB Athyreosis 5 TSHR- Familial gestational hyperthyroidism 1 TSHR Familial hyperthyroidism due to mutations in TSH receptor 1 TSHR Hypothyroidism due t o TSH receptor mutations 1 TSHR Thyroid hypoplasia 5 TSHR Microtia 1 TSHZ1 Familial exudative vitreoretinopathy 5 TSPAN12 X-linked nonsyndromic intellectual deficit 24 TSPAN7 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TSPEAR 45,X/46,XY mixed gonadal dysgenesis 2 TSPY1 Partial chromosome Y deletion 8 TSPY1 Sudden infant death - dysgenesis of the testes 1 TSPYL1 Spinocerebellar ataxia type 11 1 TTBK2 Isolated CoQ-cytochrome C reductase deficiency 6 TTC19 Infantile autosomal recessive medullary cystic kidney disease 3 TTC21B Jeune syndrome 6 TTC21B Syndromic diarrhea 2 TTC37 Multiple intestinal atresia 1 TTC7A Bardet-Biedl syndrome 17 TTC8 Retinitis pigmentosa 6 1 TTC8 Autosomal recessive limb-girdle muscular dystrophy type 2J 1 TTN

179

Table IX-21781338.1 Early-onset myopathy with fatal cardiomyopathy 1 TTN Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 TTN Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 TTN Familial isolated arrhythmogenic ventricular dysplasia, right dominant form 10 TTN Familial isolated dilated cardiomyopathy 38 TTN Hereditary proximal myopathy with early respiratory failure 1 TTN Tibial muscular dystrophy 2 TTN Ataxia with vitamin Edeficiency 1 TTPA Transthyretin-related familial amyloid cardiomyopathy 1 TTR Lissencephaly due t o TUBA1A mutation 1 TUBA1A Polymicrogyria with optic nerve hypoplasia 1 TUBA8 Autosomal dominant macrothrombocytopenia 3 TUBB1 Congenital fibrosis of extraocular muscles 4 TUBB2B Polymicrogyria due to TUBB2B mutation 1 TUBB2B Congenital fibrosis of extraocular muscles 4 TUBB3 Cortical dysgenesis with pontocerebellar hypoplasia due t o TUBB3 mutation 1 TU BB3 Hypomyelination with atrophy of basal ganglia and cerebellum 1 TUBB4A Primary dystonia, DYT4 type 1 TUBB4A Autosomal recessive chorioretinopathy - microcephaly 1 TUBGCP6 Combined oxidative phosphorylation defect type 4 1 TUFM Leber congenital amaurosis 18 TULP1 Retinitis pigmentosa 61 TULP1 Autosomal recessive npnsyndromic intellectual deficit 15 TUSC3 Isolated brachycephaly 3 TWIST1 Isolated scaphocephaly 3 TWISTl Saethre-Chotzen syndrome 4 TWIST1 Focal facial dermal dysplasia 2 TWIST2 Familial isolated dilated cardiomyopathy 38 TXN RD2 Autosomal recessive hyper IgE syndrome due to TYK2 deficiency 1 TYK2 Juvenile rheumatoid factor-negative polyarthritis 9 TYK2 Oligoarticular juvenile arthritis 9 TYK2 Mitochondrial neurogastrointestinal encephalomyopathy 3 TYMP Ocular albinism with congenital sensorineural deafness 2 TYR Oculocutaneous albinism type 1A 1 TYR Oculocutaneous albinism type I B 1 TYR Type 1 minimal pigment oculocutaneous albinism 1 TYR Type 1 temperature-sensitive oculocutaneous al binism 1 TYR Waardenburg syndrome type 2 4 TYR Nasu-Hakola disease 2 TYROBP

180

Table IX-21781338.1 Oculocutaneous albinism type 3 1 TYRP1 X-linked distal arthrogryposis multiplex congenita 1 UBA1 Behcet disease 9 UBAC2 Intellectual deficit, X-linked, Nascimento type 1 UBE2A Angelman syndrome 6 UBE3A Blepharophimosis-intellectual deficit syndrome due to UBE3B deficiency 1 UBE3B Schnyder corneal dystrophy 1 UBIAD1 Amyotrophic lateral sclerosis 23 UBQLN2 Johanson-Blizzard syndrome 1 UBR1 Early-onset progressive neurodegeneration - blindness - ataxia - spasticity 1 UCHL1 Early-onset progressive neurodegeneration - blindness - ataxia - spasticity 1 UCHL1 Young adult-onset Parkinsonism 13 UCHL1 Hyperinsulinism due to UCP2 deficiency 1 UCP2 22qll.2 deletion syndrome 7 UFD1L Crigler-Najjar syndrome type 1 1 UGT1A1 Transient familial neonatal hyperbilirubinemia 1 UGT1A1 Autosomal dominant medullary cystic kidney disease with or without hyperuricemia 2 UMOD Familial juvenile hyperuricemic nephropathy type 1 1 UMOD Hereditary orotic aciduria 1 UMPS Cone rod dystrophy 22 UNC119 Cone rod dystrophy 22 UNC119 Idiopathic CD4 lymphocytopenia 1 UNC119 Familial hemophagocytic lymphohistiocytosis 4 UNC13D Herpetic encephalitis 5 UNC93B1 Hyper-lgM syndrome type 5 1 UNG Beta-ureidopropionase deficiency 1 UPB1 X-linked intellectual deficit with marfanoid habitus 3 UPF3B X-linked nonsyndromic intellectual deficit 24 UPF3B Bilateral renal agenesis 2 UPK3A Isolated CoQ-cytochrome C reductase deficiency 6 UQCRB Isolated CoQ-cytochrome C reductase deficiency 6 UQCRC2 Isolated CoQ-cytochrome C reductase deficiency 6 UQCRQ Urocanic aciduria ' 1 UROC1 Hepatoerythropoietic porphyria 1 UROD Porphyria cutanea tarda 2 UROD Congenital erythropoietic porphyria 2 UROS Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 USH1C Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 USH1C Usher syndrome type 1 9 USH1C Usher syndrome type 1 9 USH1E

181

Table IX-21781338.1 Usher syndrome type 1 9 USH1G Usher syndrome type 1 9 USH1H Usher syndrome type 1 9 USH1K Retinitis pigmentosa 6 1 USH2A Usher syndrome type 2 4 USH2A Partial chromosome Y deletion 8 USP9Y UV-sensitive syndrome 3 UVSSA Caudal regression sequence 2 VANGL1 Cervical spina bifida aperta 6 VANGL1 Cervical spina bifida cystica 6 VANGL1 Cervicothoracic spina bifida aperta 6 VANGL1 Cervicothoracic spina bifida cystica 6 VANGL1 Familial caudal dysgenesis 1 VANGL1 Lumbosacral spina bifida aperta 6 VANGL1 Lumbosacral spina bifida cystica 6 VANGL1 Thoracolumbosacral spina bifida aperta 6 VANGL1 Thoracolumbosacral spina bifida cystica 6 VANGL1 Total spina bifida aperta 6 VANGL1 Total spina bifida cystica 6 VANGL1 Upper thoracic spina bifida aperta 6 VANGL1 Upper thoracic spina bifida cystica 6 VANGL1 Cervical spina bifida aperta 6 VANGL2 Cervical spina bifida cystica 6 VANGL2 Cervicothoracic spina bifida aperta 6 VANGL2 Cervicothoracic spina bifida cystica 6 VANGL2 Isolated anencephaly/exencephaly . 2 VANGL2 Lumbosacral spina bifida aperta 6 VANGL2 Lumbosacral spina bifida cystica 6 VANGL2 Thoracolumbosacral spina bifida aperta 6 VANGL2 Thoracolumbosacral spina bifida cystica 6 VANGL2 Total spina bifida aperta 6 VANGL2 Total spina bifida cystica 6 VANGL2 Upper thoracic spina bifida aperta 6 VANGL2 Upper thoracic spina bifida cystica 6 VANGL2 Adult-onset proximal spinal muscular atrophy, autosomal dominant 1 VAPB Amyotrophic lateral sclerosis 23 VAPB Wagner disease 1 VCAN Familial isolated dilated cardiomyopathy 38 VCL Adult-onset distal myopathy due to VCP mutation 1 CP Amyotrophic lateral sclerosis 23 VCP Inclusion body myopathy with Paget disease of bone and frontotemporal 2 VCP

182

Table IX-21781338.1 dementia Spastic paraplegia - Paget disease of bone 1 VCP Hypocalcemic vitamin D resistant rickets 1 VD Chuvash erythrocytosis 1 VHL Von Hippel-Lindau disease 1 VHL Pulverulent cataract 7 VIM Arthrogryposis - renal dysfunction - cholestasis 2 VIPAR Hereditary combined deficiency of vitamin K-dependent clotting factors 2 VKORC1 Dysequilibrium syndrome 4 VLDLR X-linked myopathy with excessive autophagy 1 VMA21 Choreoacanthocytosis 1 VPS13A Cohen syndrome 1 VPS13B Arthrogryposis - renal dysfunction - cholestasis 2 VPS33B Young adult-onset Parkinsonism 13 VPS35 Autosomal recessive spastic paraplegia type 53 1 VPS37A Pontocerebellar hypoplasia type 1 4 VRK 1 Posterior polymorphous corneal dystrophy 3 VSX1 Colobomatous microphthalmia 8 VSX2 Colobomatous microphthalmia 8 VSX2 Isolated anophthalmia - microphthalmia 5 VSX2 Microphthalmia - cataract 4 VSX2 Von Willebrand disease type 1 1 VWF Von Willebrand disease type 2A 1 VWF Von Willebrand disease type 2B 1 VWF Von.Willebrand disease type 2M 1 VWF Von Willebrand disease type 2N 1 VWF Von Willebrand disease type 3 1 VWF Wiskott-Aldrich syndrome 2 WAS X-linked severe congenital neutropenia 1 WAS X-linked thrombocytopenia with normal platelets 1 WAS Williams syndrome 17 WBSCR16 Williams syndrome 17 WBSCR22 Will iams syndrome 17 WBSCR27 Bardet-Biedl syndrome 17 WDPCP Meckel syndrome 13 WDPCP Kallmann syndrome 19 WDR11 Normosmic congenital hypogonadotropic hypogonadism 18 WDR11 Cranioectodermal dysplasia 4 WDR19 Jeune syndrome 6 WDR19 Juvenile autosomal recessive medullary cystic kidney disease 4 WDR19 Cranioectodermal dysplasia 4 WDR35

183

Table IX-21781338.1 Short rib-polydactyly syndrome, Verma-Naumoff type 3 WDR35 Beta-propeller protein-associated neurodegeneration 1 WDR45 Autosomal recessive primary microcephaly 11 WDR62 Autosomal recessive primary microcephaly 11 WDR62 Hypomaturation amelogenesis imperfecta 5 WDR72 Dysequilibrium syndrome 4 WDR81 ~ Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 WFS1 Wolfram syndrome 2 WFS1 Wolf-Hirschhorn syndrome 3 WHSC1 Wolf-Hirschhorn syndrome 3 WHSC2 Wiskott-Aldrich syndrome 2 WIPF1 Progressive pseudorheumatoid arthropathy of childhood 1 WISP3 Hereditary sensory and autonomic neuropathy type 2 4 WNK1 Pseudohypoaldosteronism type 2C 1 WNK1 Pseudohypoaldosteronism type 2B 1 WNK4 Idiopathic juvenile osteoporosis 4 WNT1 Osteogenesis imperfecta type 3 9 WNT1 Osteogenesis imperfecta type 4 7 WNT1 Autosomal recessive hypohidrotic ectodermal dysplasia 3 WNT10A Odonto-onycho-dermal dysplasia 1 WNT10A Oligodontia 10 WNT10A Schopf-Schulz-Passarge syndrome 1 WNT10A Split hand-split foot malformation 4 WNT10B Tetraamelia - multiple malformations 1 WNT3 Idiopathic juvenile osteoporosis 4 WNT3A Atypical Mayer-Rokitansky-Kuster-Hauser syndrome 1 WNT4 SERKAL syndrome 1 WNT4 Autosomal dominant Robinow syndrome 1 WNT5A Fuhrmann syndrome 1 WNT7A Phocomelia, Schinzel type 1 WNT7A Dyskeratosis congenita 10 WRAP53 Werner syndrome 1 WRN 46,XY partial gonadal dysgenesis 7 WT1 Denys-Drash syndrome 1 WT1 Denys-Drash syndrome 1 WT1 Desmoplastic small round cell tumor 2 WT1 Familial idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial sclerosis 4 WT1 Familial idiopathic steroid-resistant nephrotic syndrome with focal segmental hyalinosis 11 WT1 Frasier syndrome 1 WT1

184

Table IX-21781338.1 Meacham syndrome 1 WT1 Nephroblastoma 5 WT1 Sporadic idiopathic steroid-resistant nephrotic syndrome with diffuse mesangial sclerosis 1 WT1 WAGR syndrome 3 WT1 46,XY partial gonadal dysgenesis 7 WWOX Wieacker-Wolff syndrome 2 WWS Xanthinuria type 1 1 XDH X-linked lymphoproliferative disease 2 XIAP cLeod neuroacanthocytosis syndrome 1 XK Xeroderma pigmentosum complementation group A 1 XPA Xeroderma pigmentosum complementation group C 1 XPC Renin-angiotensin-aldosterone system-blocker-induced angioedema 1 XPNPEP2 Late-onset autosomal recessive medullary cystic kidney disease 3 XPNPEP3 Hereditary breast cancer 4 XRCC2 Autosomal dominant intermediate Charcot-Marie-Tooth disease type C 1 YARS Mitochondrial myopathy and sideroblastic anemia 2 YARS2 17pl3.3 microduplication syndrome 2 YWHAE Distal 17pl3.3 microdeletion syndrome 1 YWHAE Miller-Dieker syndrome 3 YWHAE Combined immunodeficiency due to ZAP70 deficiency 1 ZAP70 ICF syndrome 2 ZBTB24 Autosomal recessive nonsyndromic intellectual deficit 15 ZC3H14 Wieacker-Wolff syndrome 2 ZC4H2 Intellectual deficit, X-linked, Raymond type 1 ZDHHC9 X-linked intellectual deficit with marfanoid habitus 3 ZDHHC9 Fuchs endothelial corneal dystrophy 4 ZEB1 Posterior polymorphous corneal dystrophy 3 ZEB1 Mowat-Wilson syndrome due t o a point mutation 1 ZEB2 Mowat-Wilson syndrome due t o monosomy 2q22 1 ZEB2 Transient neonatal diabetes mellitus 5 ZFP57 3-methylglutaconic aciduria type 3 2 ZFP90 Congenital diaphragmatic hernia 2 ZFPM2 Tetralogy of Fallot 8 ZFPM2 Autosomal recessive spastic paraplegia type 15 1 ZFYVE26 Isolated Dandy-Walker malformation 2 ZIC1 Alo bar holoprosencephaly 14 ZIC2 Lobar holoprosencephaly 14 ZIC2 Microform holoprosencephaly 14 ZIC2 Midline interhemispheric variant of holoprosencephaly 14 ZIC2 Semilobar holoprosencephaly 14 ZIC2

185

Table IX-21781338.1 Septopreoptic holoprosencephaly 14 ZIC2 Double outlet right ventricle 3 ZIC3 Isolated congenitally uncorrected transposition of the great arteries 1 ZIC3 Situs ambiguus 6 ZIC3 Isolated Dandy-Walker malformation 2 ZIC4 Hutchinson-Gilford progeria syndrome 2 ZMPSTE24 Lethal restrictive dermopathy 2 ZMPSTE24 Mandibuloacral dysplasia with type B lipodystrophy 1 ZMPSTE24 Postaxial Polydactyly type A, bilateral 2 ZN F141 Microcephalic primordial dwarfism due to ZNF335 deficiency 1 ZN F335 Familial exudative vitreoretinopathy 5 ZNF408 X-linked nonsyndromic intellectual deficit 24 ZNF41 Joubert syndrome with oculorenal defect 6 ZNF423 Brittle cornea syndrome 2 ZN F469 Retinitis pigmentosa 61 ZNF513 CAMOS syndrome 1 ZNF592 X-linked nonsyndromic intellectual deficit 24 ZNF674 X-linked nonsyndromic intellectual deficit 24 ZN F711 Seborrhea-like dermatitis with psoriasiform elements 1 ZNF750 X-linked nonsyndromic intellectual deficit 24 ZNF81 Myxofibrosarcoma 3 Ondine syndrome 5

186

Table IX-21781338.1 CLAIMS

What is claimed is:

1. A gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VIT13, VIT14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX-1,

IX- 1A, LX-2, LX-3, XIV- 1, or Section VIII.

2. A composition, e.g., pharmaceutical composition, comprising a gRNA molecule of claim 1.

3. The composition of claim 2, further comprising a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.

4. The composition of claim 2, wherein said Cas9 molecule is an eaCas9 molecule.

5. The composition of claim 2, wherein said Cas9 molecule is an eiCas9 molecule.

6. The composition of claim 2, wherein said composition comprises a payload, e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.

7. The composition of claim 6, wherein the payload comprises: an epigenetic modifier, e.g., a molecule that modifies DNA or chromatin component, e.g., a molecule that modifies a histone, e.g., an epigenetic modifier described herein, e.g., in Section VI; a transcription factor, e.g., a transcription factor described herein, e.g., in Section VI; a transcriptional activator domain;

298 an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitor; a small molecule; an antibody; an enzyme; an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase; an a nucleic acid, e.g., an enzymatically active nucleic acid, e.g., a ribozyme, or an mRNA, siRNA, of antisense oligonucleotide.

8. The composition of claim 2, further comprising a Cas9 molecule, e.g., an eiCas9, molecule.

9. The composition of claim 7, wherein said payload is coupled, e.g., covalently or noncovalently, to a Cas9 molecule, e.g., an eiCas9 molecule.

10. The composition of claim 9, wherein said payload is coupled to said Cas9 molecule by a linker.

11. The composition of claim 10, wherein said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions.

12. The composition of claim 11, wherein said linker is, or comprises, a bond described herein, e.g., in Section XL

13. The composition of claim 10, wherein said linker is, or comprises, an ester bond.

14. The composition of claim 9, wherein said payload comprises a fusion partner fused to a Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule.

299 15. The composition of claim 7, wherein said payload is coupled, e.g., covalently or noncovalently, to the gRNA molecule.

16. The composition of claim 15, wherein said payload is coupled to said gRNA molecule by a linker.

17. The composition of claim 16, wherein said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions.

18. The composition of claim 16, wherein said linker is, or comprises, a bond described herein, e.g., in Section XL

19. The composition of claim 16, wherein said linker is, or comprises, an ester bond.

20. The composition of claim 2, comprising an eaCas9 molecule.

21. The composition of claim 2, comprising an eaCas9 molecule which forms a double stranded break in the target nucleic acid.

22. The composition of claim 2, comprising an eaCas9 molecule which forms a single stranded break in the target nucleic acid.

23. The composition of claim 22, wherein said single stranded break is formed in the complementary strand of the target nucleic acid.

24. The composition of claim 22, wherein said single stranded break is formed in the strand which is not the complementary strand of the target nucleic acid.

25. The composition of claim 22, comprising HNH-like domain cleavage activity but having no, or no significant, N-terminal RuvC-like domain cleavage activity.

300 26. The composition of claim 22, comprising N-terminal RuvC-like domain cleavage activity but having no, or no significant, HNH-like domain cleavage activity.

27. The composition of claim 21, wherein said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

28. The composition of claim 22, wherein said single stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

29. The composition of claim 20, further comprising a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

30. The composition of claim 29, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

31. The composition of claim 29, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

32. The composition of claim 29, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

33. The composition of claim 29, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:

301 a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

34. The composition of claim 3, further comprising a second gRNA molecule, e.g., a second gRNA molecule from claim 100.

35. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule mediate breaks at different sites in the target nucleic acid, e.g., flanking a target position.

36. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are complementary to the same strand of the target.

37. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are complementary to the different strands of the target.

38. The composition of claim 34, wherein said Cas9 molecule mediates a double stranded break.

39. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are configured such that first and second break made by the Cas9 molecule flank a target position.

40. The composition of claim 39, wherein said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

41. The composition of claim 40, further comprising a template nucleic acid.

42. The composition of claim 40, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

302 43. The composition of claim 42, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of a target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

44. The composition of claim 43, wherein the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

45. The composition of claim 44, wherein the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

46. The composition of claim 34, wherein said Cas9 molecule mediates a single stranded break.

47. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in the same strand of the nucleic acid target, e.g., in the case of transcribed sequence, the template strand or the non- template strand.

48. The composition of claim 47, wherein said first and second break flank a target position.

303 49. The composition of claim 48, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

50. The composition of claim 48, further comprising a template nucleic acid.

51. The composition of claim 49, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

52. The composition of claim 51, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

53. The composition of claim 51, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

54. The composition of claim 51, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-

23, VII-24, VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

304 55. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in different strands of the target.

56. The composition of claim 55, wherein said first and second break flank a target position.

57. The composition of claim 56, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

58. The composition of claim 55, further comprising a template nucleic acid.

59. The composition of claim 58, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

60. The composition of claim 58, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

61. The composition of claim 58, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

62. The composition of claim 58, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at

305 least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

63. The composition of claim 34, comprising a second Cas9 molecule.

64. The composition of claim 63, wherein one or both of said Cas9 molecule and said second Cas9 molecule are eiCas9 molecules.

65. The composition of claim 64, wherein said eiCas9 molecule is coupled to a payload by a linker and said second eiCas9 molecules is coupled to a second payload by a second linker.

66. The composition of claim 65 wherein said payload and said second payload are the same.

67. The composition of claim 65 wherein said payload and said second payload are different.

68. The composition of claim 65 wherein said linker and said second linker are the same.

69. The composition of claim 65, wherein said linker and said second linker are different, e.g., have different release properties, e.g., different release rates.

70. The composition of claim 65, wherein said payload and said second payload are each described herein, e.g., in Section VI, e.g., in Table VI- 1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.

71. The composition of claim 65, wherein said payload and said second payload can interact, e.g., they are subunits of a protein.

306 72. The composition of claim 63, wherein one of both of said Cas9 molecule and said second Cas9 molecule are eaCas9 molecules.

73. The composition of claim 72, wherein said eACas9 molecule comprises a first cleavage activity and said second eACas9 molecule comprises a second cleavage activity.

74. The composition of claim 73, wherein said cleavage activity and said second cleavage activity are the same, e.g., both are N-terminal RuvC-like domain activity or are both HNH-like domain activity.

75. The composition of claim 73 wherein said cleavage activity and said second cleavage activity are different, e.g., one is N-terminal RuvC-like domain activity and one is HNH-like domain activity.

76. The composition of claim 72, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for, e.g., NGGNG, NNAGAAW (W = A or T), or NAAR (R = A or G).

77. The composition of claim 63, wherein said Cas9 molecule and said second Cas9 molecule both mediate double stranded breaks.

78. The composition of claim 63, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.

79. The composition of claim 77, wherein said gRNA molecule and said second gRNA molecule are configured such that first and second break flank a target position.

80. The composition of claim 79, wherein, one of said first and second double stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

307 81. The composition of claim 77, further comprising a template nucleic acid.

82. The composition of claim 81, wherein the template nucleic acid a nucleotide that corresponds to a nucleotide of the target position.

83. The composition of claim 81, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ ,-2, ΓΧ -3, XIV- 1, or Section VIII.

84. The composition of claim 83, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

85. The composition of claim 83, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX-2, IX-3, XIV- 1, or Section VIII.

86. The composition of claim 77, wherein one of said Cas9 molecule and said second Cas9 molecule mediates a double stranded break and the other mediates a single stranded break.

308 87. The composition of claim 77, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.

88. The composition of claim 87, wherein said gRNA molecule and said second gRNA molecule are configured such that a first and second break flank a target position.

89. The composition of claim 88, wherein said first and second break flank a target position.

90. The composition of claim 89, wherein, one of said first and second breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

91. The composition of claim 88, further comprising a template nucleic acid.

92. The composition of claim 91, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

93. The composition of claim 91, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

94. The composition of claim 91, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

309 95. The composition of claim 91, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

96. The composition of claim 63, wherein said Cas9 molecule and said second Cas9 molecule both mediate single stranded breaks.

97. The composition of claim 96, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.

98. The composition of claim 96, wherein said first and second break flank a target position.

99. The composition of claim 96, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

100. The composition of claim 96, further comprising a template nucleic acid.

101. The composition of claim 100, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

102. The composition of claim 100, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:

310 a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

103. The composition of claim 100, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

104. The composition of claim 100, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

105. The composition of claim 96, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break are in the same strand.

106. The composition of claim 105, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.

107. The composition of claim 105, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position.

108. The composition of claim 107, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

311 109. The composition of claim 107, further comprising a template nucleic acid.

110. The composition of claim 109, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

111. The composition of claim 109, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

112. The composition of claim 109, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

113. The composition of claim 109, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

114. The composition of claim 96, wherein said first and second break are on the different strands.

312 115. The composition of claim 114, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another Pam described herein.

116. The composition of claim 114, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break are on different strands.

117. The composition of claim 114, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position.

118. The composition of claim 117, wherein said first and second break flank a target position.

119. The composition of claim 117, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

120. The composition of claim 117, further comprising a template nucleic acid.

121. The composition of claim 117, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

122. The composition of claim 117, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, IX-3, XIV- 1, or Section VIII.

123. The composition of claim 117, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in:

313 a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

124. The composition of claim 117, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

125. A composition, e.g., a pharmaceutical composition, comprising a gRNA molecule and a second gRNA molecule described herein.

126. The composition of claim 125, further comprising a nucleic acid, e.g., a DNA or mRNA, that encodes a Cas9 molecule described herein.

127. The composition of claim 125, further comprising a nucleic acid, e.g., a DNA or RNA, that encodes a second Cas9 molecule described herein.

128. The composition of claim 125, further comprising a template nucleic acid described herein.

129. A composition, e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes one or more gRNA molecules described herein.

130. The composition of claim 129, wherein said nucleic acid comprises a promoter operably linked to the sequence that encodes a gRNA molecule, e.g., a promoter described herein..

314 131. The composition of claim 129, wherein said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second gRNA molecule, e.g., a promoter described herein.

132. The composition of claim 131, wherein the promoter and second promoter are different promoters.

133. The composition of claim 131, wherein the promoter and second promoter are the same.

134. The composition of claim 129, wherein the nucleic acid further encodes a Cas9 molecule described herein.

135. The composition of claim 129, wherein the nucleic acid further encodes a second Cas9 molecule described herein.

136. The composition of claim 134, wherein said nucleic acid comprises a promoter operably linked to the sequence that encodes a Cas9 molecule, e.g., a promoter described herein.

137. The composition of claim 135, wherein said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second Cas9 molecule, e.g., a promoter described herein.

138. The composition of claim 135, wherein the promoter and second promoter are different promoters.

139. The composition of claim 135, wherein the promoter and second promoter are the same.

140. The composition of claim 129, further comprising a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.

315 141. A composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of a) a Cas9 molecule, b) a second Cas9 molecule, c) a gRNA molecule, and d) a second gRNA molecule.

142. The composition of claim 141, wherein each of a, b, c and d present are encoded on the same duplex molecule.

143. The composition of claim 141, wherein a first sequence selected from of a, b, c and d is encoded on a first duplex molecule and a second sequence selected from a, b, c, and d is encoded on a second duplex molecule.

144. The composition of claim 141, wherein said nucleic acid encodes: a and c; a, c, and d; or a, b, c, and d.

145. The composition of claim 141, further comprising a Cas9 molecule, e.g., comprising one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.

146. The composition of claim 141, further comprising an mRNA encoding Cas9 molecule, e.g., comprising one or more mRNAs encoding one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.

147. The composition of claim 141, further comprising a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.

316 148. A nucleic acid of claim 129.

149. A nucleic acid of claim 141.

150. A composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule);and c) optionally, a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.

151. A composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

152. A composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ; b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

153. A composition comprising:

317 a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

154. A method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with:

3) a composition comprising:

318 a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; or

4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

155. The method of claim 153, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by, one dosage form, mode of delivery, or formulation.

156. The method of claim 145, wherein: a) a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by, a first dosage form, a first mode of delivery, or a first formulation; and b) an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.

157. The method of claim 154, wherein the cell is an animal or plant cell.

158. The method of claim 154, wherein the cell is a mammalian, primate, or human cell.

319 159. The method of claim 154, wherein the cell is a human cell, e.g., a cell from described herein, e.g., in Section VIIA.

160. The method of claim 154, wherein the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.

161. The method of claim 154, wherein the cell is a human cell, e.g., a cancer cell or other cell characterized by a disease or disorder.

162. The method of claim 155, wherein the target nucleic acid is a chromosomal nucleic acid.

163. The method of claim 155, wherein the target nucleic acid is an organellar nucleic acid.

164. The method of claim 155, wherein the target nucleic acid is a mitochondrial nucleic acid.

165. The method of claim 155, wherein the target nucleic acid is a chloroplast nucleic acid.

166. The method of claim 155, wherein the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

167. The method of claim 155, wherein the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

168. The method of claim 155, wherein said method comprises: modulating the expression of a gene or inactivating a disease organism.

320 169. The method of claim 155, wherein said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.

170. The method of claim 155, wherein said cell is a cell characterized by an unwanted genomic component, e.g., a viral genomic component.

171. The method of claim 155, wherein the cell is a cell described herein, e.g., in Section IIA.

172. The method of claim 155, wherein a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.

173. The method of claim 169, wherein the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor

174. The method of claim 155, comprising cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

175. The method of claim 155, wherein said composition comprises a template nucleic acid.

176. The composition of claim 155, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

177. The composition of claim 155, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

321 178. The composition of claim 155, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

179. The method of claim 155, wherein, a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted; c) the coding sequence of a gene is cleaved; d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected; e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon;

180. The method of claim 155, wherein a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.

181. A method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of:

1) a composition comprising:

322 a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ; b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;

3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; or

4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and

323 c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.

182. The method of claim 181, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.

183. The method of claim 181, wherein: a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, in a first mode of delivery, or first formulation; and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.

184. The method of claim 181, wherein the subject is an animal or plant.

185. The method of claim 181, wherein the subject is a mammalian, primate, or human.

186. The method of claim 154, wherein the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section VIIA.

187. The method of claim 181, wherein the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.

188. The method of claim 181, wherein the target nucleic acid is a chromosomal nucleic acid.

189. The method of claim 181, wherein the target nucleic acid is an organellar nucleic acid.

190. The method of claim 181, wherein the nucleic acid is a mitochondrial nucleic acid.

324 191. The method of claim 181, wherein the nucleic acid is a chloroplast nucleic acid.

192. The method of claim 181, wherein the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

193. The method of claim 181, wherein said method comprises modulating expression of a gene or inactivating a disease organism.

194. The method of claim 181, wherein the target nucleic acid is the nucleic acid of a cell characterized by unwanted proliferation, e.g., a cancer cell.

195. The method of claim 181, wherein said target nucleic acid comprises an unwanted genomic component, e.g., a viral genomic component.

196. The method of claim 181, wherein a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.

197. The method of claim 181, wherein the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.

198. The method of claim 181, comprising cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.

199. The method of claim 181, wherein said composition comprises a template nucleic acid.

200. The composition of claim 181, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.

325 201. The composition of claim 181, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

202. The composition of claim 181, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

203. The composition of claim 181, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:

204. The method of claim 181, wherein, a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted; c) the coding sequence of a gene is cleaved; d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected; e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon;

326 205. The method of claim 181, wherein a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.

206. A composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule.

207. A composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule.

208. A composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule.

327 209. A composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA ,encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.

210. A method of delivering a payload to a cell, e.g., by targeting a payload to target nucleic acid, comprising contacting said cell with:

328 3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; or

4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA ,encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.

211. The method of claim 206, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.

212. The method of claim 206, wherein: a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, first mode of delivery, or first formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.

213. The method of claim 206, wherein the cell is an animal or plant cell.

214. The method of claim 206, wherein the cell is a mammalian, primate, or human cell.

329 215. The method of claim 206, wherein the cell is a human cell, e.g., a human cell described herein, e.g., in Section VIIA.

216. The method of claim 206, wherein the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.

217. The method of claim 206, wherein the cell is a human cell, e.g., a cancer cell, a cell comprising an unwanted genetic element, e.g., all or part of a viral genome.

218. The method of claim 206, wherein the gRNA mediates targeting of a chromosomal nucleic acid.

219. The method of claim 206, wherein the gRNA mediates targeting of a selected genomic signature.

220. The method of claim 206, wherein the gRNA mediates targeting of an organellar nucleic acid.

221. The method of claim 206, wherein gRNA mediates targeting of a mitochondrial nucleic acid.

222. The method of claim 206, wherein the gRNA mediates targeting of a chloroplast nucleic acid.

223. The method of claim 206, wherein the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

224. The method of claim 206, wherein the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

330 225. The method of claim 206, wherein the payload comprises a payload described herein, e.g., in Section VI.

226. The method of claim 206, wherein said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.

227. The method of claim 206, wherein said cell is characterized by an unwanted genomic component, e.g., a viral genomic component.

228. The method of claim 206, wherein a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.

229. The method of claim 206, wherein the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.

230. The method of claim 206, wherein the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or tumor suppressor.

231. The method of claim 206, wherein the gRNA targets a cancer cell, e.g., a cancer cell disclosed herein, e.g., in Section VIIA.

232. The method of claim 206, wherein the gRNA targets a cell which has been infected with a virus.

233. A method of treating a subject, e.g., by targeting a payload to target nucleic acid, comprising administering to the subject, an effective amount of:

1) a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);

331 b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule;

3) a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; or

4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA ,encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.

332 234. The method of claim 233, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.

235. The method of claim 233, wherein: a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage, mode of delivery form or formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, mode of delivery, or formulation.

236. The method of claim 233, wherein the subject is an animal or plant cell.

237. The method of claim 233, wherein the subject is a mammalian, primate, or human cell.

238. The method of claim 233, wherein the gRNA mediates targeting of a human cell, e.g., a human cell described herein, e.g., in Section VIIA.

239. The method of claim 233, wherein the gRNA mediates targeting of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.

240. The method of claim 233, wherein the gRNA mediates targeting of a cancer cell or a cell comprising an unwanted genomic element, e.g., all or part of a viral genome.

241. The method of claim 233, wherein the gRNA mediates targeting of a chromosomal nucleic acid.

242. The method of claim 233, wherein the gRNA mediates targeting of a selected genomic signature.

333 243. The method of claim 233, wherein the gRNA mediates targeting of an organellar nucleic acid.

244. The method of claim 233, wherein gRNA mediates targeting of a mitochondrial nucleic acid.

245. The method of claim 233, wherein the gRNA mediates targeting of a chloroplast nucleic acid.

246. The method of claim 233, wherein the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.

247. The method of claim 233, wherein the gRNA targets a cell characterized by unwanted proliferation, e.g., a cancer cell, e.g., a cancer cell from Section VIIA, e.g., from Table VII-11.

248. The method of claim 233, wherein the gRNA targets a cell characterized by an unwanted genomic component, e.g., a viral genomic component.

249. The method of claim 233, wherein a control element, e.g., a promoter or enhancer, is targeted.

250. The method of claim 233, wherein the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.

251. The method of claim 233, wherein the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.

334 252. The method of claim 233, wherein the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.

253. The method of claim 233, wherein the gRNA targets a cancer cell.

254. The method of claim 233, wherein the gRNA targets a cell which has been infected with a virus.

255. The method of claim 233, wherein at least one eaCas9 molecule and a payload are administered.

256. The method of claim 233, wherein the payload comprises a payload described herein, e.g., in Section VI.

257. A reaction mixture comprising a composition described herein and a cell.

258. The composition of claim 155, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24,

VII-25, IX- 1, IX- 1A, ΓΧ -2, ΓΧ -3, XIV- 1, or Section VIII.

335