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WO 2015/071474 A2 21 May 2015 (21.05.2015) P O P C T

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WO 2015/071474 A2 21 May 2015 (21.05.2015) 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 2015/071474 A2 21 May 2015 (21.05.2015) P O P C T (51) International Patent Classification: Krzysztof; Simmeringer Hauptstrasse 45/8, A-1 110 Vi C12N 15/11 (2006.01) enna (AT). FONFARA, Ines; Helmstedter Strasse 144, 38102 Braunschweig (DE). (21) International Application Number: PCT/EP2014/074813 (74) Agent: PILKINGTON, Stephanie Joan; Potter Clarkson LLP, The Belgrave Centre, Talbot Street, Nottingham NG1 (22) International Filing Date: 5GG (GB). 17 November 2014 (17.1 1.2014) (81) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of national protection available): AE, AG, AL, AM, (26) Publication Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (30) Priority Data: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, 61/905,835 18 November 2013 (18. 11.2013) US HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (71) Applicant: CRISPR THERAPEUTICS AG [CH/CH]; KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, Aeschenvorstadt 36, CH-4051 Basel (CH). MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (72) Inventors: CHARPENTIER, Emmanuelle; Boeckler- SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, strasse 18, 38102 Braunschweig (DE). CHYLINSKI, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. [Continued on nextpage] (54) Title: CRISPR-CAS SYSTEM MATERIALS AND METHODS (57) Abstract: The invention relates to Type II CRIS- PR-Cas systems of Cas9 enzymes, guide RNAs and associ S- pyogenes . s ated specific PAMs. ft e m pW s * S. iher p i s " C. jejuni P. a N meningitidis . Gvi ia Acn * c 9 or ol g ¾ ¾- ¾ < < 4 nt 3 l II © Figure 4 (84) Designated States (unless otherwise indicated, for every SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, kind of regional protection available): ARIPO (BW, GH, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, Published: TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, — without international search report and to be republished DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, upon receipt of that report (Rule 48.2(g)) LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, — with sequence listing part of description (Rule 5.2(a)) CRISPR-CAS SYSTEM MATERIALS AND METHODS Field of the Invention [0001] The invention relates to type II CRISPR-Cas systems of Cas9 enzymes, guide RNAs and associated specific PAMs. This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 61/905,835 filed November 18, 2013, which is incorporated by reference herein in its entirety. Incorporation by Reference of the Sequence Listing [0002] This application contains, as a separate part of disclosure, a Sequence Listing in computer- readable form (filename: 481 28_SeqListing.txt; 7,869,256 bytes - ASCII text file; created November 14 , 201 4) which is incorporated by reference herein in its entirety. Background [0003] Editing genomes using the RNA-guided DNA targeting principle of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) immunity has been exploited widely over the past few months ( 1- 13). The main advantage provided by the bacterial type II CRISPR-Cas system lies in the minimal requirement for programmable DNA interference: an endonuclease, Cas9, guided by a customizable dual-RNA structure ( 14). As initially demonstrated in the original type II system of Streptococcus pyogenes, trans-activating CRISPR RNA (tracrRNA) ( 15 ,16) binds to the invariable repeats of precursor CRISPR RNA (pre-crRNA) forming a dual-RNA (14-1 7) that is essential for both RNA co-maturation by RNase III in the presence of Cas9 ( 15-1 7), and invading DNA cleavage by Cas9 (14,1 5,17-1 9). As demonstrated in Streptococcus, Cas9 guided by the duplex formed between mature activating tracrRNA and targeting crRNA (14-16) introduces site-specific double- stranded DNA (dsDNA) breaks in the invading cognate DNA ( 14 , 7-1 9). Cas9 is a multi-domain enzyme ( 14,20,21 ) that uses an HNH nuclease domain to cleave the target strand (defined as complementary to the spacer sequence of crRNA) and a RuvC-like domain to cleave the non-target strand (14,22,23), enabling the conversion of the dsDNA cleaving Cas9 into a nickase by selective motif inactivation (2,8, 14,24,25). DNA cleavage specificity is determined by two parameters: the variable, spacer-derived sequence of crRNA targeting the protospacer sequence (a protospacer is defined as the sequence on the DNA target that is complementary to the spacer of crRNA) and a short sequence, the Protospacer Adjacent Motif (PAM), located immediately downstream of the protospacer on the non-target DNA strand ( 14,18,23,26-28). [0004] Recent studies have demonstrated that RNA-guided Cas9 can be employed as an efficient genome editing tool in human cells ( 1 ,2,8, 11), mice (9, 10), zebrafish (6), drosophila (5), worms (4), plants ( 12, 3), yeast (3) and bacteria (7). The system is versatile, enabling multiplex genome engineering by programming Cas9 to edit several sites in a genome simultaneously by simply using multiple guide RNAs (2,7,8, 10). The easy conversion of Cas9 into a nickase was shown to facilitate homology-directed repair in mammalian genomes with reduced mutagenic activity (2,8,24,25). In addition, the DNA-binding activity of a Cas9 catalytic inactive mutant has been exploited to engineer RNA-programmable transcriptional silencing and activating devices (29,30). [0005] To date, RNA-guided Cas9 from S. pyogenes, Streptococcus thermophilus.Neisseria meningitidis and Treponema denticola have been described as tools for genome manipulation ( 1- 13,24,25,31 -34 and Esvelt et al. PMID: 24076762). Summary [0006] The present invention expands the RNA-programmable Cas9 toolbox to additional orthologous systems. The diversity and interchangeability of dual-RNA:Cas9 in eight representatives of phylogenetically defined type II CRISPR-Cas groups was examined herein. The results of this work not only introduce a wider range of Cas9 enzymes, guide RNA structures and associated specific PAMs but also enlighten the evolutionary aspects of type II CRISPR-Cas systems, including coevolution and horizontal transfer of the system components. [0007] In an aspect, the present disclosure provides guide RNAs, both single-molecule and double- molecule guide RNAs, as well as methods for manipulating DNA in a cell using the guide RNAs and/or DNAs (including vectors) encoding the guide RNAs. Complexes comprising the guide RNAs and Cas9 endonucleases are also provided [0008] In some embodiments, the single-molecule guide RNAs comprise a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5 or wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5. In some embodiments, the protein-binding segment comprises a CRISPR repeat set out in Supplementary Table S5 that is the CRISPR repeat cognate to the tracrRNA of the protein-binding segment. In some embodiments, the DNA-targeting segment comprises RNA complementary to a protospacer-like sequence in a target DNA 5' to a PAM sequence. In some embodiments, the tracrRNA and CRISPR repeat are respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA. In some embodiments, the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA. [0009] In some embodiments, the single-molecule guide RNA comprises a sequence that hybridizes to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701 . [0010] n another aspect, the disclosure provides a DNA encoding a single-molecule guide RNA of the invention. [0011] n yet another aspect, the disclosure provides a vector comprising a DNA encoding a single- molecule guide RNA of the invention. [0012] In still another aspect, the disclosure provides a cell comprising a DNA encoding a single- molecule guide RNA of the invention. [0013] In an aspect, the disclosure provides a double-molecule guide RNA comprising: a targeter- RNA and an activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5 or wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5. In some embodiments, the double-molecule guide RNA comprises a modified backbone, a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, a base modification, a modification or sequence that provides for modified or regulated stability, a modification or sequence that provides for subcellular tracking, a modification or sequence that provides for tracking, or a modification or sequence that provides for a binding site for a protein or protein complex. In some embodiments, the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5. In some embodiments, the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5 that is the cognate CRISPR repeat of the tracrRNA of the activator-RNA.
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