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WO 2014/172470 A2 23 October 2014 (23.10.2014) P O P C T

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WO 2014/172470 A2 23 October 2014 (23.10.2014) 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 2014/172470 A2 23 October 2014 (23.10.2014) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/85 (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/US2014/034387 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, 16 April 2014 (16.04.2014) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (30) Priority Data: ZW. 61/812,720 16 April 2013 (16.04.2013) US 61/824,920 17 May 2013 (17.05.2013) US (84) Designated States (unless otherwise indicated, for every 61/858,437 25 July 2013 (25.07.2013) US kind of regional protection available): ARIPO (BW, GH, 61/865,888 14 August 2013 (14.08.2013) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (71) Applicant: WHITEHEAD INSTITUTE FOR BIOMED¬ TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, ICAL RESEARCH [US/US]; Nine Cambridge Center, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, LV, Cambridge, MA 02142 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors: JAENISCH, Rudolf; Nine Cambridge Center, KM, ML, MR, NE, SN, TD, TG). Cambridge, MA 02142 (US). WANG, Haoyi; Nine Cam bridge Center, Cambridge, MA 02142 (US). YANG, Hui; Published: Nine Cambridge Center, Cambridge, MA 02142 (US). — without international search report and to be republished SHFVALILA, Chikdu; Nine Cambridge Center, Cam upon receipt of that report (Rule 48.2(g)) bridge, MA 02142 (US). CHENG, Wu, Albert; Nine Cambridge Center, Cambridge, MA 02142 (US). (74) Agents: COLLINS, Anne, J. et al; Hamilton, Brook, Smith & Reynolds, P.C., 530 Virginia Rd, P.O. Box 9133, Concord, MA 01742-9133 (US). < © (54) Title: METHODS OF MUTATING, MODIFYING OR MODULATING NUCLEIC ACID IN A CELL OR NONHUMAN MAMMAL (57) Abstract: The invention is directed to a method of mutating one or more target nucleic acid sequences in a stem cell or a zygote comprising introducing into the stem cell or zygote (i) ribonucleic acid (RNA) sequences that comprise a portion that is comple - mentary to a portion of each of the target nucleic acid sequences and comprise a binding site for a CRISPR associated (Cas) protein; and a Cas nucleic acid sequence or a variant thereof that encodes a Cas protein having nuclease activity. The stem cell or zygote is maintained under conditions in which the target nucleic acid sequences are mutated in the stem cell or zygote. The invention is also directed to methods of producing a non human mammal carrying mutations and methods of modulating the expression and/or activ ity target nucleic acid sequences and cells or zygotes. METHODS OF MUTATING, MODIFYING OR MODULATING NUCLEIC ACID IN A CELL OR NONHUMAN MAMMAL RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 61/8 12,720, filed on April 16, 2013; U.S. Provisional Application No. 61/824,920, filed on May 17, 2013; U.S. Provisional Application No. 61/858,437, filed on July 25, 2013; and U.S. Provisional Application No. 61/865,888, filed on August 14, 2013. [0002] The entire teachings of the above applications are incorporated herein by reference. GOVERNMENT SUPPORT [0003] This invention was made with government support under HD 045022 and R37CA084198 from the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION [0004] Genetically modified mice represent a crucial tool for understanding gene function in development and disease. Mutant mice are conventionally generated by insertional mutagenesis (Copeland and Jenkins, 2010; Kool and Berns, 2009) or by gene targeting methods (Capecchi, 2005). In conventional gene targeting methods, mutations are introduced through homologous recombination in mouse embryonic stem (ES) cells. Targeted ES cells injected into wild-type blastocysts can contribute to the germline of chimeric animals, generating mice containing the targeted gene modification (Capecchi, 2005). It is costly and time- consuming to produce single gene knockout mice, and even more so to make double mutant mice. Moreover, in most other mammalian species no established ES cell lines are available that contribute efficiently to chimeric animals, which greatly limits the genetic studies in many species. [0005] Alternative methods have been developed to accelerate the process of genome modification by directly injecting DNA or mRNA of site-specific nucleases into the one cell embryo to generate DNA double strand break (DSB) at a specified locus in various species (Bogdanove and Voytas, 201 1; Carroll et al, 2008; Urnov et al, 2010). DSBs induced by these site-specific nucleases can then be repaired by either error-prone non-homologous end joining (NHEJ) resulting in mutant mice and rats carrying deletions or insertions at the cut site (Carbery et al, 2010; Geurts et al, 2009; Sung et al, 2013; Tesson et al, 201 1). If a donor plasmid with homology to the ends flanking the DSB is co-injected, high-fidelity homologous recombination can produce animals with targeted integrations (Cui et al., 201 1; Meyer et al., 2010). Because these methods require the complex designs of zinc finger nucleases (ZNFs) or Transcription activator-like effector nucleases (TALENs) for each target gene and because the efficiency of targeting may vary substantially, no multiplexed gene targeting has been reported to date. [0006] Thus, improved methods for producing genetically modified non-human mammals, such as mice, are needed. SUMMARY OF THE INVENTION [0007] Described herein is the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins (CRISPR/Cas) system to drive both non-homologous end joining (NHEJ) based gene disruption and homology directed repair (HDR) based precise gene editing to achieve highly efficient and simultaneous targeting of multiple nucleic acid sequences in cells and nonhuman mammals. [0008] Accordingly, in one aspect, the invention is directed to a method of mutating one or more target nucleic acid sequences in a (one or more) stem cell or a zygote comprising introducing into the stem cell or zygote (i) one or more ribonucleic acid (RNA) sequences that comprise a portion that is complementary to a portion of each of the one or more target nucleic acid sequences and comprise a binding site for a CRISPR associated (Cas) protein; and a Cas nucleic acid sequence or a variant thereof that encodes a Cas protein having nuclease activity. The stem cell or zygote is maintained under conditions in which the one or more RNA sequences hybridize to the portion of each of the one or more target nucleic acid sequences, and the Cas protein cleaves each of the one or more target nucleic acid sequences upon hybridization of the one or more RNA sequences to the portion of the target nucleic acid sequence, thereby mutating one or more target nucleic acid sequences in the stem cell or zygote. [0009] In some aspects, the invention is directed to a method of producing a nonhuman mammal carrying mutations in one or more target nucleic acid sequences comprising introducing into a zygote or an embryo (i) one or more ribonucleic acid (RNA) sequences that comprise a portion that is complementary to a portion of each of the one or more target nucleic acid sequences and comprise a binding site for a CRISPR associated (Cas) protein; and ii) a Cas nucleic acid sequence or a variant thereof that encodes a Cas protein having nuclease activity. The zygote or the embryo is maintained under conditions in which RNA hybridizes to the portion of each of the one or more target nucleic acid sequences, and the Cas protein cleaves each of the one or more target nucleic acid sequences upon hybridization of the RNA to the portion of the target nucleic acid sequence, thereby producing an embryo having one or more mutated nucleic acid sequences. The embryo having one or more mutated nucleic acid sequences may be transferred into a foster nonhuman mammalian mother. The foster nonhuman mammalian mother is maintained under conditions in which one or more offspring carrying the one or more mutated nucleic acid sequences are produced, thereby producing a nonhuman mammal carrying mutations in one or more target nucleic acid sequences. [0010] In some aspects, the invention is directed to a method of modulating the expression and/or activity of one or more target nucleic acid sequences in one or more cells or zygotes comprising introducing into the cell or zygote (i) one or more ribonucleic acid (RNA) sequences that comprise a portion that is complementary to each of the one or more target nucleic acid sequences and comprise a binding site for a CRISPR associate (Cas) protein; (ii) a Cas nucleic acid sequence or a variant thereof that encodes the Cas protein that targets but does not cleave the target nucleic acid sequence; and (iii) an effector domain.
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