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

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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). < 00 © o (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.
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