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WO 2018/035387 Al 22 February 2018 (22.02.2018) W ! P O PCT

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WO 2018/035387 Al 22 February 2018 (22.02.2018) W ! P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization III International Bureau (10) International Publication Number (43) International Publication Date WO 2018/035387 Al 22 February 2018 (22.02.2018) W ! P O PCT (51) International Patent Classification: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, C12N 15/10 (2006.01) C12N 15/85 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12N 15/63 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US2017/047458 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, 17 August 2017 (17.08.2017) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 62/376,372 17 August 2016 (17.08.2016) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, 62/437,03 1 20 December 2016 (20.12.2016) US 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, (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 415 Main Street, Cambridge, MA 02142 (US). KM, ML, MR, NE, SN, TD, TG). MASSACHUSETTS INSTITUTE OF TECHNOLO¬ GY [US/US]; 77 Massachusetts Avenue, Cambridge, MA Published: 02139 (US). — with international search report (Art. 21(3)) (72) Inventors: ZHANG, Feng; c/o The Broad Institute, Inc., 415 Main Street, Cambridge, MA 02142 (US). SCOTT, David, Arthur; c/o Massachusetts Institute of Technolo gy, 77 Massachusetts Avenue, Cambridge, MA 02 139 (US). YAN, Winston, Xia; c/o Massachusetts Institute of Tech nology, 77 Massachusetts Avenue, Cambridge, MA 02139 (US). CHOUDHURY, Sourav; c/o The Broad Institute, Inc., 415 Main Street, Cambridge, MA 02142 (US). HEI- DENREICH, Matthias; c/o The Broad Institute, Inc., 415 Main Street, Cambridge, MA 02142 (US). (74) Agent: NIX, F., Brent; Johnson, Marcou & Isaacs, LLC, 27 City Square, Suite 1, Hoschton, GA 30548 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, = (54) Title: NOVEL CRISPR ENZYMES AND SYSTEMS =Ξ (57) Abstract: In one aspect, embodiments disclosed herein are directed to FIG 1 engineered CRISPR-Cas effector proteins that comprise at least one modifi- — cation compared to an unmodified CRISPR-Cas effector protein that enhances _ binding of the of the CRISPR complex to the binding site and/or alters edit- ing preference as compared to wild type. In certain example embodiments, target e CRISPR-Cas effector proteins a Type II effector protein. In certain other " ¾ , example embodiments, the Type V effector protein is Cas9 or an orthologs , , p r ' o engineered variant thereof Example Cas9 proteins suitable for use in the ¾ ¾ embodiments disclosed herein are discussed in further detail below. (SpCas9) tarqel variation vsrisnts «« * TG o PAH - RNA guide (SpCas9) NOVEL CRISPR ENZYMES AND SYSTEMS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/376,372 filed August 17, 2016, and U.S. Provisional Application No. 62/437,031 filed December 20, 2016, STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under grant numbers MH100706 and MH1 10049 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION [0003] The present invention generally relates to systems, methods and compositions related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. The present invention also generally relates to delivery of large payloads and includes novel delivery particles, particularly using lipid and viral particle, and also novel viral capsids, both suitable to deliver large payloads, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR protein (e.g., Cas, Cas9), CRISPR-Cas or CRISPR system or CRISPR-Cas complex, components thereof, nucleic acid molecules, e.g., vectors, involving the same and uses of all of the foregoing, amongst other aspects. Additonally, the present invention relates to methods for developing or designing CRISPR-Cas system based therapy or therapeutics. BACKGROUND OF THE INVENTION [0004] 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 new 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. [0005] 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 is no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cas 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. [0006] The development of CRISPR-Cas RNA-guided endonucleases for eukaryotic genome editing has sparked intense interest in the use of this technology for therapeutic applications. [0007] Extensive research has led to the identification of different technologies which can address the challenges of safety and efficacy. In order to allow the translation of this genome editing technologies to the clinic. There is a need for the development of an algorithm for developing a CRISPR-Cas based therapeutic, which takes into account the different variables which need to be considered. [0008] In contrast to small molecule therapies, which target highly conserved protein active sites, treatment of disease at the genomic level must contend with significant levels of genetic variation in patient populations. Recently, large scale sequencing datasets from the Exome Aggregation Consortium (ExAC) and 1000 Genomes Project have provided an unprecedented view of the landscape of human genetic variation. This variation can affect both the efficacy of a CRISPR-based therapeutic, by disrupting the target site, and its safety, by generating off-target candidate sites. [0009] 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 [0010] In certain example embodiments, an engineered CRISPR-Cas effector protein that complexes with a nucleic acid comprising a guide sequence to form a CRISPR complex, and wherein in the CRISPR complex the nucleic acid molecule target one or more polynucleotide loci and the protein comprises at least one modification compared to the unmodified protein that enhances binding of the CRISPR complex to the binding site and/or alters editing preferences as compared to wildtype. The editing preference may relate to indel formation. In certain example embodiments, the at least one modification may increase formation of one or more specific indels at a target locus. The CRISPR-Cas effector protein may be Type II CRISPR-Cas effector protein. In certain example embodiments, the CRISPR-Cas protein is Cas9 or orthologue thereof. [0011] In certain other example embodimnets, the invention is directed to vectors for delivery of the CRISPR-Cas system, including vector based systems allowing for encoding of both the effector protein and guide sequence in a single vector. [0012] In certain other example embodimnets, the invention relates to methods for developing or designing CRISPR-Cas systems. In an aspect, the present invention relates to methods for developing or designing CRISPR-Cas system based therapy or therapeutics. The present invention in particular relates to methods for improving CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics. Key characteristics of successful CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics involve high specificity, high efficacy, and high safety. High specificity and high safety can be achieved among others by reduction of off-target effects. [0013] The methods of the present invention in particular involve optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, as described herein further elsewhere.
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