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WO 2018/005873 Al 0 4 January 2018 (04.01.2018) W !P O PCT

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WO 2018/005873 Al 0 4 January 2018 (04.01.2018) 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 2018/005873 Al 0 4 January 2018 (04.01.2018) W !P O PCT (51) International Patent Classification: BASUDEB, Maji [US/US]; 7 1 Elm Street, Apt. 2, Cam C12N 15/11 (2006.01) bridge, MA 01239 (US). ZETSCHE, Bernd [DE/US]; 383 Washington Street, Gloucester, MA 01930 (US). ZHANG, (21) International Application Number: Feng [US/US]; 100 Pacific Street, Apt. 11, Cambridge, MA PCT/US201 7/0401 15 02139 (US). (22) International Filing Date: (74) Agent: CASSON, Lawrence, P. et al; Vedder Price P.C., 29 June 2017 (29.06.2017) 1633 Broadway, New York, NY 10019 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Language: 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, 62/356,028 29 June 2016 (29.06.2016) 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, JO, JP, KE, KG, KH, KN, KP, 415 Main Street, Cambridge, MA 02142 (US). KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MASSACHUSETTS INSTITUTE OF TECHNOLOGY MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, [US/US]; 77 Massachusetts Ave., Cambridge, MA 02139 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (US). THE BRIGHAM AND WOMEN'S HOSPITAL, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, INC. [US/US]; 75 Francis Street, Boston, MA 021 15 (US). TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (72) Inventors; and (84) Designated States (unless otherwise indicated, for every (71) Applicants: CHOUDHARY, Amit [US/US]; 89 Trow kind of regional protection available): ARIPO (BW, GH, bridge Street, Apt. 6, Cambridge, MA 01238 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (54) Title: CRISPR-CAS SYSTEMS HAVING DESTABILIZATION DOMAIN -TVPDHFR PP7 VP64: , .„ . — +T ;.DHFR¾PP VP64 — - ♦ l N dCas9 Proteasomal degradation 3 1 IL1RN CD > < 10 > - + RFP PP7 DHFR.PP7 00 Fig. 13A ©o (57) Abstract: The disclosure includes non-naturally occurring or engineered DNA-or RNA-guided nuclease systems, comprising 00 guide-binding adaptors each associated with at least one destabilization domain (DD), along with compositions, systems and complexes o involving such systems, nucleic acid molecules and vectors encoding the same, delivery systems involving the same, uses therefor. o [Continued on next page] W O 2018/005873 A l Illlll II lllll lllll lllll ill III III Hill Hill lllll lllll lllll llll III llll llll 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: CRISPR-CAS SYSTEMS HA VING DESTABILIZA TION DOMAINS RELATED APPLICATIONS AND INCORPORATION BY REFERENCE [0001] This application claims the benefit of US Provisional Patent Application No. 62/356,028, filed June 29, 2017. [0002] Each of these patents, patent publications, and applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, together with any instructions, descriptions, product specifications, and product sheets for any products mentioned therein are incorporated by reference herein, and may be employed in the practice of the invention. Moreover, all documents cited or referenced herein ("herein cited documents"), and all 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. 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 [0003] This invention was made with government support under grant nos. MH100706 and MH1 10049 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION [0004] The present invention generally relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR enzyme or effector (e.g. Cas9, Cpfl, C2cl, C2c2, C2c3, Group 29/30, Casl3) and variants thereof, 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. BACKGROUND OF THE INVENTION [0005] 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. [0006] RNA-guided endonucleases, such as Cas9, are easily targeted to any desired DNA or RNA locus using guide RNAs (gRNA), ushering in a slew of transformative technologies. For example, Cas9 has enabled facile and efficient induction of genomic alterations in cells and multiple organisms, and Cas9-based gene drives permit super-Mendelian self-propagation of such modifications (3). Furthermore, catalytically inactive CRISPR effectors, such as Cas9 (dCas9) can be fused to a wide range of effectors, including fluorescent proteins for genome imaging (4), enzymes that modify DNA or histones for epigenome editing (5), and transcription regulating domains for controlling endogenous gene expression (6). [0007] Despite such advances, a critical need still exists for methods to precisely and switchably regulate CRISPR effector activities across multiple dimensions, including dose, target, and time (7). Finely-tuned control of CRISPR effector proteins levels is important, as high concentrations result in elevated off-target DNA cleavage. Rapid disabling of activity after a desired genomic modification is also essential (8), but the genome editing toolkit currently lacks highly transportable and modular methodologies that can be applied with minimal optimization to diverse RNA-guided nucleases. In the context of gene regulation, such as by dead CRISPR effector-based transcriptional activators, dose control of transcript induction is critical to ensure that physiologically relevant levels of gene expression are induced. Similarly useful would be the abilities to rapidly reverse transcript induction and to control the expression of multiple genes with orthogonal inducers. [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 sequence targeting with a wide array of applications. This invention addresses this need and provides related advantages. The CRISPR/Cas or the CRISPR-Cas system (both terms may be used interchangeably throughout this application) does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target, in other words the Cas enzyme can be recruited to a specific DNA target using said short RNA molecule. It will be appreciated that reference herein to the Cas protein includes any type of CRISPR/Cas system effector protein, such as without limitation Cas9, Cpfl, C2cl, C2c2, C2c3, Casl3, group 29/30, as well as variants or modified effectors, as also described herein elsewhere (e.g. catalytically inactive variants, nickases, variants having modified activity, stability and/or specificity, variants having altered PAM recognition, split effectors, etc.). As an alternative to the CRISPR/Cas system, the present invention also envisages the use of argonaute systems, as for instance detailed in Gao et al. (2016) "DNA-guided genome editing using the Natronobacterium gregoryi Argonaute" Nat Biotechnol. 2016 May 2 . doi: 10.1038/nbt.3547. [Epub ahead of print]. It will be understood that whenever reference is made herein to CRISPR/Cas proteins or systems, such is equally applicable to argonautes. As a corollary, reference to a guide, guide polynucleotide, guide RNA (gRNA) may include guide RNA or guide DNA in the context of argonautes. [0010] Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering and optimization of these genome engineering tools, which are aspects of the claimed invention. In some embodiments, the terms 'CRISPR enzyme' and 'nucleic acid-targeting effector protein' may be used interchangeably.
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