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Wo2019/135816 ( (51) International Patent Classification: GOPADHYAY, Soumyashree Ashok [US/US]; c/o 75 A61K 48/00 (2006.01) Francis Street, Boston, Massachusetts 021 15 (US). (21) International Application Number: (74) Agent: RUTLEDGE, Rachel D. et al.; Johnson, Marcou & PCT/US20 18/057 182 Isaacs, LLC, P. O .Box 691, Floschton, Georgia 30548 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 23 October 2018 (23. 10.2018) kind of national protection av ailable) . AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (25) Filing Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (26) Publication Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (30) Priority Data: KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 62/575,948 23 October 2017 (23. 10.2017) US MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 62/765,347 20 August 2018 (20.08.2018) US OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 415 Main Street, Cambridge, Massachusetts 02142 (US). TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. THE BRIGHAM AND WOMEN'S HOSPITAL, INC. (84) Designated States (unless otherwise indicated, for every [US/US]; 75 Francis Street, Boston, Massachusetts 021 15 kind of regional protection available) . ARIPO (BW, GH, (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (72) Inventors; and UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (71) Applicants: CHOUDHARY, Amit [US/US]; c/o 75 Fran¬ TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, cis Street, Boston, Massachusetts 021 15 (US). LIM, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Donghyun [US/US]; c/o 415 Main Street, Cambridge, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Massachusetts 02142 (US). COX, Kurt [US/US]; c/o 415 TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, Main Street, Cambridge, Massachusetts 02142 (US). GAN- KM, ML, MR, NE, SN, TD, TG). (54) Title: NOVEL NUCLEIC ACID MODIFIERS HDR NHEJ activator inhibitor FIG. 1 (57) Abstract: The present inventions generally relate to site-specific delivery of nucleic acid modifiers and includes novel DNA- binding proteins and effectors that can be rapidly programmed to make site-specific DNA modifications. The present inventions also provide a synthetic all-in-one genome editor (SAGE) systems comprising designer DNA sequence readers and a set of small molecules that induce double-strand breaks, enhance cellular permeability, inhibit NHEJ and activate HDR, as well as methods of using and delivering such systems. [Continued on next page] ||| ||||| ||||| ||||| |||| 11| ||| ||||| ||||| ||||| ||||| ||||| |||| limn nil nil nil Published: without international search report and to be republished upon receipt of that report (Rule 48.2(g)) with sequence listing part of description (Rule 5.2(a)) NOVEL NUCLEIC ACID MODIFIERS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/575,948, filed October 23, 2017, and U.S. Provisional Application No. 62/765,347 filed August 20, 2018. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. STATEMENT AS TO FEDERALLY FUNDED RESEARCH [0002] This invention was made with government support under contract number N66001-17- 2-4055 awarded by the Defense Advanced Research Projects Agency, Grant No. W91 1NF1610586 awarded by the Army Research Office, and Grant No. AI126239 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0003] The contents of the electronic sequence listing (BROD-2770WP.ST25.txt”; Size is 4 Kilobytes and it was created on October 23, 2018) is herein incorporated by reference in its entirety. TECHNICAL FIELD [0004] The present inventions generally relate to nucleic acid modifiers with novel DNA readers and effectors that can be rapidly programmed to make site-specific DNA modifications. Among other aspects, the DNA readers provide features of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR proteins (e.g., Cas, Cas9, Cpfl, C2cl, Casl3 and the like), CRISPR-Cas or CRISPR systems or CRISPR-Cas complexes, components thereof, peptide nucleic acid (PNA), nucleic acid molecules, vectors, involving the same and uses of all of the foregoing. BACKGROUND [0005] Of the three elements of central dogma (Proteins, RNA, and DNA), nearly all therapeutic agents target proteins. Genome and RNA editing have ushered in an era where DNA and RNA can be potential targets, expanding the scope of therapeutic targets to both the coding and non-coding regions of the genome. However, the agents used to accomplish genome editing do not display attributes of a typical therapeutic agent, and in many cases, the activity of these agents are described as genome vandalism rather than genome editing. As such, there is much room to expand the repertoire of genome editors. [0006] There is an urgent need to develop countermeasures that will rectify any accidental or malevolent genomic alterations in an organism or population. Currently, such genomic alterations can be corrected using CRISPR-nucleases. Following induction of double-strand breaks by the nuclease, most cells adopt Non-Homologous End Joining (NHEJ) repair pathway over Homology Directed Repair (HDR), resulting in random insertions and deletions (indels). The in vivo applications face additional challenges. This state-of-art of genomic remediation method is unsuitable for countermeasures for multiples reasons. First, HDR pathways are poorly activated in most cells and nearly always out-competed by non-homologous end joining (NHEJ) resulting in imprecise, error-prone, and uncontrolled genetic alterations. Second, efficient cellular delivery of CRISPR-nucleases requires the development of paradigm shifting delivery technologies that are currently unavailable. Third, owing to delivery issues, multiplex genomic remediation on a scale to match the genomic diversity present in a population is not feasible. Fourth, CRISPR-based systems are sensitive to proteases/nucleases, limiting their use in multiple countermeasure scenarios (e.g., ecosystem setting). Fifth, the slow kinetics of CRISPR-system will be problematic from a countermeasure perspective, where often fast genomic remediation is desired. Sixth, the components of the CRISPR-system (i.e., a large protein and RNA) potentially will be difficult and expensive to mass-produce. [0007] CRISPR-Cas9 from S. pyogenes was evolved for rapid and efficient destruction of phage DNA but lacks the functionalities needed for precision genome edits. Following a double-strand break in a knock-in experiment, an exogenously supplied single-stranded oligo donor (ssODN) is integrated at the break site. This integration can be facilitated if the ssODN is readily available at the break site. Further, local inhibition of the NHEJ pathway and/or local activation of HDR at the strand-break site can also tip the balance in favor of DNA recombination. Several small- molecule inhibitors of NHEJ pathway and HDR activators have been reported. However, the mutagenicity and toxicity of genome-wide NHEJ inhibition or HDR activation severely limit the utility of such molecules. There is also a need to improve homology-directed repair (HDR) efficiency. Increased efficiency of repair is highly desirable in disease models and therapies. [0008] 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. There remains a need for new genome engineering technologies. [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 [0010] The present disclosure provides a synthetic all-in-one genome editor (SAGE) comprising designer DNA sequence readers and a set of small molecules that induce double- strand breaks and enhance DNA repair by homology directed repair (HDR). [0011] In one aspect, the disclosure relates to an engineered, non-naturally occurring nucleic acid modifying system, comprising: (a) an engineered, non-naturally occurring CRISPR/Cas protein; (b) a guide nucleic acid, wherein the guide nucleic acid directs sequence specific binding of the CRISPR/Cas protein to a target nucleic acid; and (c) one or more effector components, wherein the one or more effector components facilitate DNA repair by homology directed repair (HDR). [0012] In certain embodiments, the one or more effector components comprise one or more single-stranded oligo donors (ssODNs). In certain embodiments, the one or more effector components comprise one or more NHEJ inhibitors. In certain embodiments, the one or more effector components comprise one or more HDR activators. In certain embodiments, the one or more effector components comprise a single-stranded oligo donor (ssODN), one or more NHEJ inhibitors, and one or more HDR activators. [0013] The CRISPR/Cas protein can be selected from the group consisting of an engineered Cas9, Cpfl, Casl2b, Casl2c, Casl3a, Casl3b, Casl3c, and Casl3d protein. In certain embodiments, the CRISPR/Cas protein is an engineered Cas9 protein. The CRISPR/Cas protein can comprise one or more engineered cysteine amino acids. In a preferred embodiment, the CRISPR/Cas protein is an SpCas9 protein comprising C80S and C574S mutations and one or more mutations selected from the group consisting of M1C, S204C, D435C, E532C, Q674C, Q826C, S867C, E945C, S1025C, E1026C, N1054C, E1068C, S 1116C, K 1153C, E1207C. In certain embodiments, the CRISPR/Cas protein comprises a sortase recognition sequence Leu- Pro-Xxx-Thr-Gly.
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