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WO 2017/184786 Al 26 October 2017 (26.10.2017) W !P O PCT

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WO 2017/184786 Al 26 October 2017 (26.10.2017) W !P O PCT (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 2017/184786 Al 26 October 2017 (26.10.2017) W !P O PCT (51) International Patent Classification: (US). YAN, Winston [US/US]; 145 Longwood Ave., Apt. C12N 15/11 (2006.01) C12N 15/113 (2010.01) 1, Brookline, MA 02446 (US). SANJANA, Neville, Es- C12N 9/22 (2006.01) C12N 15/63 (2006.01) pi [US/US]; 4 Washington Square Village, Apt. 16L, New York, NY 10012 (US). JONES, Sara [US/US]; c/o The (21) International Application Number: Broad Institute Inc., 415 Main Street, Cambridge, MA PCT/US20 17/028461 02142 (US). (22) International Filing Date: (74) Agent: KOWALSKI, Thomas, J. et al; Vedder Price P.C., 19 April 2017 (19.04.2017) 1633 Broadway, New York, NY 10019 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Langi English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/324,834 19 April 2016 (19.04.2016) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (71) Applicants: THE BROAD INSTITUTE INC. [US/US]; HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, 415 Main Street, Cambridge, MA 02142 (US). KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MASSACHUSETTS INSTITUTE OF TECHNOLOGY MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, [US/US]; 77 Massachusetts Ave., Cambridge, MA 02139 PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (US). 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. (72) Inventors; and (71) Applicants: ZHANG, Feng [US/US]; 100 Pacific Street, (84) Designated States (unless otherwise indicated, for every Apt. 11, Cambridge, MA 02139 (US). ZETSCHE, Bemd kind of regional protection available): ARIPO (BW, GH, [DE/US]; 383 Washington Street, Gloucester, MA 01930 GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (54) Title: CPF1 COMPLEXES WITH REDUCED INDEL ACTIVITY \ V PA V PAM P A ¾ 00 00 Fig.. (57) Abstract: The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein o and at least one targeting nucleic acid component like a guide RNA. [Continued on nextpage] WO 2017/184786 Al llll II II 11III I II I II I II III I II III II I II UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 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, LV, 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, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) CPF1 COMPLEXES WITH REDUCED INDEL ACTIVITY RELATED APPLICATIONS AND INCORPORATION BY REFERENCE [0001] This application claims benefit of and priority to U.S. Provisional Application No. 62/324,834, filed April 19, 2016, incorporated herein by reference. [0002] Reference is made to U.S. Provisional Application Nos. 62/324,777 and 62/324,820 filed April 19, 2016, U.S. Provisional Application No. 62/351,558 filed June 17, 2016, U.S. Provisional Application No. 62/360,765 filed July 11, 2016, U.S. Provisional Application No. 62/376,379 filed August 17, 2016, and U.S. Provisional Application Nos 62/410,196 and 62/410,240, filed October 19, 2016, herein incorporated by reference. [0003] The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and ail documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0004] This invention was made with government support under MH1 00706, MH1 10049, and HG008171 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION [0005] The present invention generally relates to systems, methods and compositions used for the control of gene expression involving sequence targeting, such as perturbation of gene transcripts or nucleic acid editing, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. BACKGROUND OF THE INVENTION [0006] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for ew genome engineering technologies that employ novel strategies and molecular mechanisms and are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. This would provide a major resource for new applications in genome engineering and biotechnology. [0007] The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR-Cas system loci has more than 50 gene families and there s no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cos gene identification of about 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture. A new classification of CRISPR-Cas systems is proposed in which these systems are broadly divided into two classes, Class 1 with multisubunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein. Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools and the prediction of putative novel effector proteins and their engineering and optimization is important. [0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE INVENTION [0009] There exists a pressing need for alternative and robust systems and techniques for targeting nucleic acids or polynucleotides (e.g. DNA or any hybrid or derivative thereof) with a wide array of applications. This invention addresses this need and provides related advantages. Adding the novel DNA-targeting systems of the present application to the repertoire of genomic and epigenomic targeting technologies may transform the study and perturbation or editing of specific target sites through direct detection, analysis and manipulation. To utilize the DNA- targeting systems of the present application effectively for genomic or epigenomic targeting without deleterious effects, it is critical to understand aspects of engineering and optimization of these DNA targeting tools. [0010] The invention provides a method of modifying sequences associated with or at a target locus of interest, the method comprising delivering to said locus a no --naturally occurring or engineered composition comprising a Type V CRISPR-Cas loci effector protein and one or more nucleic acid components, wherein the effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the sequences associated with or at the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break. In a preferred embodiment, the sequences associated with or at the target locus of interest comprises DNA and the effector protein is encoded by a subtype V-A CRISPR-Cas loci or a subtype V-B CRISPR-Cas loci. [00 ] It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPR protein Cas protein and CRISPR Cas are generally used interchangeably and at ail points of reference herein refer by analogy to novel CRISPR effector proteins further described in this application, unless othenvise apparent, such as by specific reference to Cas9. The CRISPR effector proteins described herein are preferably Cpf 1 effector proteins. [0012] The invention provides a method of modifying sequences associated with or at a target locus of interest, the method comprising delivering to said sequences associated with or at the locus a non-naturally occurring or engineered composition comprising a Cpfl loci effector protein and one or more nucleic acid components, wherein the Cpfl effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the sequences associated with or at the target locus of interest.
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