RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C § 119(e) to U.S. provisional application, U.S. S.N. 62/469,219, filed March 9, 2017, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Tumor- specific immune responses may be elicited by peptides generated from proteins expressed in tumor cells or on tumor cell surface (e.g., tumor- specific antigens). Native peptides derived from tumor- specific antigens are tolerated as "self by the immune system and do not elicit strong immune response against the tumor- specific antigen. Altered versions of the native peptides derived from tumor- specific antigens (e.g., heteroclitic peptides or cryptic peptides) may be engineered to elicit potent immune reactions through the MHC-I and MHC-II antigen presentation pathways, which also produce cross-reactive responses towards the native tumor- specific antigen sequences.

[0003] It is well established that the immune system can function to kill tumor cells, including both primary and metastatic cancer cells. Indeed, evidence that the immune system recognizes the presence of neoplastic cancerous cells is supported by the existence of infiltrating lymphocytes in tumor tissues (Haskill et ah, 1978, Contemp. Top. Immunobiol. 8: 107-170; Vose and Moore, 1985, Semin. Hematol. 22: 27-40). Yet, for reasons that are not completely clear, despite the presence of immune cells, tumors often prevail and not only survive but metastasize to distant sites with unrestricted growth. Recent advances in the understanding of T cell activation and recognition of target cells have begun to permit some progress in development of T cell mediated cancer immunotherapy (Schwartz, 1992, Cell ! I: 1065-1068; Pardoll, 1992, Curr. Opin. Immunol. 4: 619-623).

SUMMARY OF THE INVENTION

[0004] Described herein are systems, methods, compositions, and kits for producing immunogenic peptides derived from tumor specific antigens (e.g., heteroclitic epitopes or cryptic epitopes) that may be used as cancer vaccines in vivo or ex vivo. Targeted mutations are introduced into tumor- specific antigens using gene editing agents, e.g., a nucleobase editor comprising a programmable DNA binding domain (e.g., catalytically-inactive Cas9 or a Cas9 nickase) fused to a cytosine deaminase, to generate altered versions of peptides arising from the tumor- specific antigens (heteroclitic epitopes) or peptides arising from normally untranslated regions of the tumor- specific antigen genes (cryptic peptides). The heteroclitic peptides or cryptic peptides may be generated in vivo in a subject (e.g., a subject who has cancer) and presented to the adaptive immune system via the MHC class I or MHC class II pathway, which in turn induces a strong adaptive immune response, e.g. , T cell response and B cell response. Such an adaptive immune response is antigen specific and is effective in reducing tumor growth and preventing metastasis.

[0005] Some aspects of the present disclosure provide methods of eliciting a tumor- specific immune response in a subject in need thereof, the methods including administering to the subject a therapeutically effective amount of a composition comprising: (i) a fusion protein comprising (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a cytosine deaminase domain; and (ii) a guide nucleotide sequence, wherein the guide nucleotide sequence of (ii) targets the fusion protein of (i) to a polynucleotide encoding a tumor- specific antigen in a tumor cell, wherein the fusion protein changes a target cytosine (C) base to a thymine (T) base via deamination.

[0006] In some embodiments, the polynucleotide comprises a coding strand and a complementary strand. In some embodiments, the polynucleotide comprises a coding region and a non-coding region. In some embodiments, the polynucleotide encoding the tumor- specific antigen is located in the genome of the tumor cell. In some embodiments, deamination of the target C base results in a C-G base-pair to thymine- adenine (T-A) base- pair change.

[0007] In some embodiments, the guide nucleotide sequence-programmable DNA binding protein domain is selected from the group consisting of: nuclease inactive Cas9 (dCas9) domains, nuclease inactive Cpfl domains, nuclease inactive Argonaute domains, and variants thereof.

[0008] In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain is a nuclease inactive Cas9 (dCas9) domain. In some embodiments, the amino acid sequence of the dCas9 domain comprises mutations corresponding to a D10A and/or H840A mutation in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the dCas9 domain comprises a mutation corresponding to a D10A mutation in SEQ ID NO: 1, and wherein the dCas9 domain comprises a histidine at the position corresponding to amino acid 840 of SEQ ID NO: 1.

[0009] In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises a nuclease inactive Cpfl (dCpfl) domain. In some embodiments, the dCpf 1 domain is from a species of Acidaminococcus or Lachnospiraceae. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises a nuclease inactive Argonaute (dAgo) domain. In some embodiments, the (dAgo) domain is from Natronobacterium gregoryi (dNgAgo).

[0010] In some embodiments, the cytosine deaminase domain comprises an apolipoprotein B niRNA-editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase is selected from the group consisting of APOBEC 1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G deaminase,

APOBEC3H deaminase, APOBEC4 deaminase, and activation-induced deaminase (AID). In some embodiments, the cytosine deaminase comprises an amino acid sequence of any of SEQ ID NOs: 27-292, 303, and 1072-1083.

[0011] In some embodiments, the fusion protein of (a) further comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the cytosine deaminase domain is fused to the N-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain. In some embodiments, the UGI domain is fused to the C-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain. In some

embodiments, the cytosine deaminase and the guide nucleotide sequence-programmable DNA-binding protein domain is fused via an optional linker. In some embodiments, the UGI domain is fused to the guide nucleotide sequence-programmable DNA-binding protein domain via an optional linker.

[0012] In some embodiments, the fusion protein comprises the structure NH ₂ -[cytosine deaminase domain] -[optional linker sequence] -[ guide nucleotide sequence-programmable DNA-binding protein domain] -[optional linker sequence] -[UGI domain] -COOH.

[0013] In some embodiments, the optional linker comprises (GGGS) _n , (SEQ ID NO: 337) (GGGGS) _n (SEQ ID NO: 308), (G)n (SEQ ID NO: 783), (EAAAK) _n (SEQ ID NO: 309), (GGS)„ (SEQ ID NO: 784), SGSETPGTSESATPES (SEQ ID NO: 310), or (XP)n (SEQ ID NO: 785) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30 and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 310). In some embodiments, the linker is (GGS) _n (SEQ ID NO: 784), and wherein n is 1, 3, or 7.

[0014] In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 293-302, 1071, and 1084.

[0015] In some embodiments, the tumor specific antigen is selected from the group consisting of: CEA; gplOO; Pmell7; mammaglobin-A; Melan-A; MART- 1 ; NY-BR-1; ERBB2; OA1; PAP; PSA; RAB38; NY-MEL- 1; TRP-1; gp75; TRP-2; tyrosinase; WT1; CD33; BAGE-1; D393-CD20n; Cyclin-Al; GAGE-1,2,8; GAGE-3,4,5,6,7; GnTVf; HERV- K-MEL; KK-LC-1; KM-HN-1; LAGE-1; LY6K; MAGE-A1; MAGE-A2; MAGE- A3;

MAGE-A4; MAGE-A6; MAGE-A9; MAGE-A10; MAGE-A12m; MAGE-C1; MAGE-C2; mucink; NA88-A; NY-ESO-1; LAGE-2; SAGE; Spl7; SSX-2; SSX-4; survivin; BIRC5; TAG- 1 ; TAG-2; TRAG-3; TRP2-INT2g; XAGE-lb; GAGED2a; BCR-ABL (b3a2);

adipophilin; AIM-2; ALDH1A1; BCLX(L); BING-4; CALCA; CD45; CD274; CPSF; cyclin Dl; DKKl; ENAH (hMena); EpCAM; EphA3; EZH2; FGF5; glypican-3; G250; MN; CAIX; HER-2; neu; HLA-DOB; Hepsin; IDOl; IGF2B3; IL13Ralpha2; Intestinal carboxyl esterase; alpha-foetoprotein; Kallikrein 4; KIF20A; Lengsin; M-CSF; MCSP; mdm-2; Meloe;

Midkine; MMP-2; MMP-7; MUC1; MUC5AC; p53; PAX5; PBF; PRAME; PSMA; RAGE- 1; RGS5; RhoC; RNF43; RU2AS; secernin 1; SOX10; STEAP1; Telomerase; TPBG; and VEGF.

[0016] In some embodiments, the target C base is in a target codon located in a coding region of the polynucleotide encoding the tumor- specific antigen. In some embodiments, the target codon is any one of the target codons in Tables 4 and 8.

[0017] In some embodiments, the target codon is converted to a modified codon selected from any one of the modified codons in Table 4. In some embodiments, the target C base is located in a non-coding region of the polynucleotide encoding the tumor specific antigen. In some embodiments, the target C base is located in an intron in the polynucleotide encoding the tumor specific antigen.

[0018] In some embodiments, the methods described herein further comprising generating an immunogenic peptide from the tumor- specific antigen. In some embodiments, the immunogenic peptide is a heteroclitic epitope. In some embodiments, the heteroclitic epitope is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, or more immunogenic than a native epitope from the tumor specific antigen. In some embodiments, the immunogenic peptide is a cryptic epitope. In some embodiments, the cryptic epitope is at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, or more immunogenic than a native epitope from the tumor specific antigen.

[0019] In some embodiments, the immunogenic peptide is displayed on the surface of the tumor cell via the MHC class I antigen presentation pathway. In some embodiments, the immunogenic peptide is displayed on the surface of an antigen presenting cell (APC) via the MHC class II antigen presentation pathway.

[0020] In some embodiments, the method is carried out in vivo. In some embodiments, the method is carried out ex vivo.

[0021] In some embodiments, the APC is selected from the group consisting of: tumor cells, dendritic cells, mononuclear phagocytes, thymic epithelial cells, and B cells.

[0022] In some embodiments, the immunogenic peptide elicits adaptive immune response against the tumor- specific antigen. In some embodiments, the adaptive immune response comprises promoting the maturation of dendritic cells, activation of CD4+T lymphocytes, activation of CD8+ T lymphocytes, activation and maturation of B lymphocytes, and/or production of tumor antigen-specific antibodies. In some embodiments, the adaptive immune response kills tumor cells, reduces tumor size, and/or prevents metastasis.

[0023] In some embodiments, the guide nucleotide sequence is an RNA. In some embodiments, the RNA is chemically modified.

[0024] In some embodiments, the guide nucleotide sequence is a single strand DNA (ssDNA).

[0025] In some embodiments, the tumor specific antigen is gplOO. In some embodiments, the gplOO is from melanoma. In some embodiments, the deamination of the target C base in codon T210 of gplOO results in a T210I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of IIDQVPFSV (SEQ ID NO: 786) is generated, and wherein the I at position 2 corresponds to the T210I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 724 and 870-888.

[0026] In some embodiments, the deamination of the target C base in codon A288 of gplOO results in a A288V mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of YLEPGPVTV (SEQ ID NO: 818) is generated, and wherein the V at position 7 corresponds to the A288V mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 725 and 889.

[0027] In some embodiments, the deamination of the target C base in codon T155 of gplOO results in a T155I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of KIWGQYWQV (SEQ ID NO: 787) is generated, and wherein the I at position 2 corresponds to the T155I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 726 and 890-892.

[0028] In some embodiments, the tumor specific antigen is melanoma antigen recognized by T cells 1 (MART-1). In some embodiments, the MART-1 antigen is from melanoma. In some embodiments, the deamination of the target C base in codon A27 of MART-1 results in a A27V mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of EVAGIGILTV (SEQ ID NO: 819) is generated, and wherein the V at position 2 corresponds to the A27V mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 727 and 893-896.

[0029] In some embodiments, the tumor specific antigen is cancer/testis antigen IB (NY- ESO-1). In some embodiments, the NY-ESO-1 antigen is from melanoma or breast cancer. In some embodiments, the deamination of the target C base in codon C165 of NY-ESO-1 results in a C165Y mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of SLLMWITQY (SEQ ID NO: 788) is generated, and wherein the C at position 9 corresponds to the C165Y mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 728 and 897.

[0030] In some embodiments, the tumor specific antigen is Tyrosinase (TYR). In some embodiments, the TYR antigen is from melanoma. In some embodiments, the deamination of the target C base in codon T373 of TYR results in a T373I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of YMNGIMSQV (SEQ ID NO: 789) is generated, and wherein the I at position 5 corresponds to the T373I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 729 and 898-901.

[0031] In some embodiments, the tumor specific antigen is tyrosinase-related protein 1 (TyRPl). In some embodiments, the TyRPl antigen is from melanoma. In some

embodiments, the deamination of the target C base in codon C244 of TyRPl results in a C244Y mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of DAEKYDICTDEY (SEQ ID NO: 790) is generated, and wherein the Y at position 5 corresponds to the C244Y mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 730 and 902. [0032] In some embodiments, the tumor specific antigen is Survivin. In some embodiments, the Survivin is from melanoma, breast cancer, or leukemia. In some embodiments, the deamination of the target C base in codon T97 of Survivin results in a T97I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of ELILGEFLKL (SEQ ID NO: 791) is generated, and wherein the I at position 3

corresponds to the T97I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 731 and 903.

[0033] In some embodiments, the tumor specific antigen is telomerase reverse

transcriptase (hTERT). In some embodiments, the hTERT is from breast cancer. In some embodiments, the deamination of the target C base in codon M549 of hTERT results in a M549I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of ILAKFLHWLI (SEQ ID NO: 792) is generated, and wherein the I at position 10 corresponds to the M549I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 735 and 916-920.

[0034] In some embodiments, the tumor specific antigen is human epidermal growth factor receptor 2 (HER2). In some embodiments, the HER2 is from breast cancer. In some embodiments, the deamination of the target C base in codon V658 of HER2 results in a V658M mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of AMVGILLVVV (SEQ ID NO: 793) is generated, and wherein the M at position 2 corresponds to the V658M mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 732 and 904-909.

[0035] In some embodiments, the deamination of the target C base in codon T912 of HER2 results in a T912I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of IIWELMTFGA (SEQ ID NO: 794) is generated, and wherein the

V at position 2 corresponds to the T912I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 733 and 910-912.

[0036] In some embodiments, the deamination of the target C base in codon A920 of HER2 results in a A920V mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of ITWELMTFGV (SEQ ID NO: 795) is generated, and wherein the

V at position 10 corresponds to the A920V mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 734 and 913-915.

[0037] In some embodiments, the tumor specific antigen is CD33. In some embodiments, the CD33 is from leukemia. In some embodiments, the deamination of the target C base in codon A65 of CD33 results in a A65V mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of VIISGDSPV (SEQ ID NO: 796) is generated, and wherein the V at position 1 corresponds to the A65V mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 740 and 930-932.

[0038] In some embodiments, the tumor specific antigen is Synovial Sarcoma X

Breakpoint 2 (SSX2). In some embodiments, the deamination of the target C base in codon A42 of SSX2 results in a A42V mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of KVSEKIFYV (SEQ ID NO: 797) is generated, and wherein the V at position 2 corresponds to the A42V mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 737 and 921.

[0039] In some embodiments, the tumor specific antigen is Wilm's tumor 1 (WT1) protein. In some embodiments, the WT1 is from leukemia. In some embodiments, the deamination of the target C base in codon C235 of WT1 results in a C235Y mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of

YMTWNQMNL (SEQ ID NO: 798) is generated, and wherein the Y at position 1 corresponds to the C235Y mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 738 and 922-925.

[0040] In some embodiments, the deamination of the target C base in codon M236 of WT1 results in a M236I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of CITWNQMNL (SEQ ID NO: 799) is generated, and wherein the I at position 2 corresponds to the M236I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 739 and 926-929.

[0041] In some embodiments, the tumor specific antigen is Epithelial cell adhesion molecule precursor (EpCAM). In some embodiments, the deamination of the target C base in codon T192 of EpCAM results in a T192I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of ILYENNVII (SEQ ID NO: 800) is generated, and wherein the I at position 9 corresponds to the T192I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 741 and 933-934.

[0042] In some embodiments, the tumor specific antigen is carcinoembryonic antigen- related cell adhesion molecules (CEA-CAM). In some embodiments, the CEA-CAM is from colorectal cancer, lung cancer, or breast cancer. In some embodiments, the deamination of the target C base in codon T314 of CEA-CAM results in a T314I mutation. In some

embodiments, a heteroclitic epitope comprising the amino acid sequence of LLTFWNPPI (SEQ ID NO: 801) is generated, and wherein the I at position 9 corresponds to the T314I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 742 and 935-936.

[0043] In some embodiments, the deamination of the target C base in codon T311 of CEA-CAM results in a T31 II mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of RITVTTITV (SEQ ID NO: 802) is generated, and wherein the V at position 2 corresponds to the T31 II mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 743 and 937-940.

[0044] In some embodiments, the deamination of the target C base in codon T688 of CEA-CAM results in a T688V mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of AVVGIMIGV (SEQ ID NO: 803) is generated, and wherein the V at position 2 corresponds to the T688V mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 744 and 941-947.

[0045] In some embodiments, the deamination of the target C base in codon V695 of CEA-CAM results in a V695M mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of IMIGMLVGV (SEQ ID NO: 804) is generated, and wherein the M at position 5 corresponds to the V695M mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 745 and 948-953.

[0046] In some embodiments, the tumor specific antigen is melanoma-associated antigen A3 (MAGEA3). In some embodiments, the deamination of the target C base in codon HI 18 of MAGEA3 results in a HI 18Y mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of KVAELVYFL (SEQ ID NO: 805) is generated, and wherein the Y at position 7 corresponds to the HI 18Y mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 746 and 954.

[0047] In some embodiments, the tumor specific antigen is melanoma-associated antigen (MAGE) common antigen A3, Al, A4, A2, or A12. In some embodiments, the deamination of the target C base in codon C181 of MAGE common antigen A3, Al, A4, A2, or

A12results in a C181Y mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of YLGLSYDGLL (SEQ ID NO: 806) is generated, and wherein the Y at position 1 corresponds to the C181Y mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 747-750 and 955-983.

[0048] In some embodiments, the tumor specific antigen is MUC-1. In some

embodiments, the deamination of the target C base in codon T93 of MUC-1 results in a T93I mutation. In some embodiments, a heteroclitic epitope comprising the amino acid sequence of AIWGQDVTSV (SEQ ID NO: 807) is generated, and wherein the I at position 2 corresponds to the T93I mutation. In some embodiments, the guide nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 751 and 984-985.

[0049] In some embodiments, the target C base is located in intron 4 of the

premelanosome protein (PMEL) gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of VYFFLPDHL (SEQ ID NO: 808). In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 752-753 and 986-998.

[0050] In some embodiments, the target C base is located on the complementary strand of open reading frame 1 (ORF1) of TYRP1 gene. In some embodiments, the target C base is located in the complementary strain of the first start codon (ATG) of ORF1 of the TYRP1 gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of MSLQRQFLR (SEQ ID NO: 809). In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 754 and 999-1005.

[0051] In some embodiments, the target C base is located on the complementary strand of the last base of intron 2 of the mannosyl (alpha- l,6-)-glycoprotein beta-l,6-N-acetyl glucosaminyltransferase (MGAT5) gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of VLPDVFIRCV (SEQ ID NO: 810). In some embodiments, the cryptic peptide is translated from exon 3 of the MGAT5 gene. In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 755 and 1006-1008.

[0052] In some embodiments, the target C base is located in open reading frame 1 (ORF1) of cancer/testis antigen 2 (LAGE-1) gene. In some embodiments, the target C base is located in the complementary strand of the first start codon of ORF1 of the LAGE-1 gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of selected from the group consisting of: MLMAQEALAFL (SEQ ID NO: 811), LAAQERRVPR (SEQ ID NO: 812), APRGVRMAV (SEQ ID NO: 813),

QGAMLAAQERRVPRAAEVPR (SEQ ID NO: 814), and

CLSRRPWKRSWSAGSCPGMPHL (SEQ ID NO: 815). In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 756 and 1009-1014.

[0053] In some embodiments, the target C base is located in intron 2 of tyrosinase-related protein 2 (TRP-2) gene. In some embodiments, the target C base is located on the

complementary strand of the first base of intron 2 of the TRP-2 gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of EVISCKLIKR (SEQ ID NO: 816). In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 757-758 and 1015-1023.

[0054] In some embodiments, the target C base is located in intron 2 of baculoviral IAP repeat containing 5 (BIRC5) gene. In some embodiments, the target C base is located on the spliceosome branch site of intron 2 of the BIRC5 gene. In some embodiments, the target C base is located in the complementary strand of the last base of intron 2 of the BIRC5 gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of AYACNTSTL (SEQ ID NO: 817). In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 759 and 1024-1029.

[0055] In some embodiments, the target C base is located in intron 1 acceptor site of BCR/ABL fusion proteins (BCR/ABL-OOF) gene. In some embodiments, the target C base is located in intron 2 acceptor site of BCR/ABL fusion proteins (BCR/ABL-OOF) gene. In some embodiments, the deamination of the target C base results in a cryptic peptide comprising the amino acid sequence of any one of SSKALQRPV (SEQ ID NO: 603), GFKQSSKAL (SEQ ID NO: 604), and ATGFKQS S KALQRPVAS (SEQ ID NO: 605). In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 761 and 1032-1045. In some embodiments, the guide nucleotide sequence comprises a nucleotide selected from the group consisting of SEQ ID NOs: 762 and 1046-1056.

[0056] In some embodiments, the methods further comprising administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor inhibits binding of CTLA-4, PD-1, PD-L1, TEVI3, LAG3, B7-H3, B7-H4, BTLA, GAL9, Chkl, or A2aR to a cognate binding partner. In some embodiments, the immune checkpoint inhibitor is an antibody or a fragment thereof. In some embodiments, the antibody is selected from anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-TIM3 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-Chkl antibodies, and anti-A2aR antibodies. In some embodiments, the antibody is selected from

pembrolizumab, nivolumab, and ipilimumab.

[0057] In some embodiments, the immune checkpoint inhibitor is a small molecule.

[0058] In some embodiments, the immune checkpoint inhibitor is a recombinant protein.

[0059] In some embodiments, the immune checkpoint inhibitor is a nucleic acid ap tamer.

[0060] In some embodiments, the immune checkpoint inhibition is performed by genome editing of a gene selected from the group consisting of: CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3, B7-H4, BTLA, GAL9, Chkl, or A2aR.

[0061] Other aspects of the present disclosure provide methods of treating cancer, the methods including administering to a subject in need thereof a therapeutically effective amount of a composition comprising: (i) a fusion protein comprising (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a cytosine deaminase domain; and (ii) a guide nucleotide sequence; wherein the fusion protein of (i) and the guide nucleotide sequence of (ii) enters a tumor cell, and wherein the guide nucleotide sequence targets the fusion protein of (i) to a polynucleotide encoding a tumor- specific antigen, wherein the fusion protein changes a target cytosine (C) residue to a (T) residue in the polynucleotide.

[0062] In some embodiments, the methods include administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor.

[0063] Further provided herein are methods of inducing a tumor- specific immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising: (i) a fusion protein comprising (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a nuclease domain; and (ii) a guide nucleotide sequence; wherein the fusion protein of (i) and the guide nucleotide sequence of (ii) enters the tumor cell, and wherein the guide nucleotide sequence targets the fusion protein of (i) to a polynucleotide encoding a tumor- specific antigen, wherein the fusion protein introduces an indel in the polynucleotide. In some embodiments, the nuclease is a Fokl nuclease.

[0064] Further provided herein are methods of inducing a tumor- specific immune response in a subject in need thereof, the methods including administering to the subject a therapeutically effective amount of a composition comprising: (i) a guide nucleotide sequence-programmable nuclease; and (ii) a guide nucleotide sequence; wherein the fusion protein of (i) and the guide nucleotide sequence of (ii) enters the tumor cell, and wherein the guide nucleotide sequence targets the fusion protein of (i) to a polynucleotide encoding a tumor- specific antigen, wherein the guide nucleotide sequence-programmable nuclease introduces an indel in the polynucleotide.

[0065] In some embodiments, the guide nucleotide sequence-programmable nuclease comprises a Cas9, a Cpfl, an Argonaute, or a variant thereof. In some embodiments, the indel causes a mutation or frame shift.

[0066] Method of inducing a tumor- specific immune response in a subject in need thereof are also provided, the methods including administering to a subject in need thereof a therapeutically effective amount of a composition comprising a fusion protein comprising (a) a programmable DNA-binding protein domain; and (b) a deaminase domain; wherein the fusion protein enters the tumor cell and changes a target base in the polynucleotide via deamination.

[0067] In some embodiments, the deaminase domain comprises a cytosine deaminase and the target base is a cytosine (C) base. In some embodiments, the programmable DNA-binding domain comprises a zinc finger nuclease (ZFN). In some embodiments, the programmable DNA-binding domain comprises a transcription activator- like effector (TALE).

[0068] In some embodiments, the programmable DNA-binding domain is a guide nucleotide sequence-programmable DNA binding protein domain. In some embodiments, the programmable DNA-binding domain is selected from the group consisting of: nuclease- inactive Cas9 domains, nuclease inactive Cpfl domains, nuclease inactive Argonaute domains, and variants thereof. In some embodiments, the programmable DNA-binding domain is associated with a guide nucleotide sequence. In some embodiments, the

deamination of the target C base results in a C to thymine (T) change. In some embodiments, the deamination of the target C base results in a C-G base pair to thymine-adenine (T-A) change in a translated codon, resulting in the incorporation of a different amino acid in an immunogenic or heteroclitic peptide. In some embodiments, the deamination of the target C base results in a C-G basepair to thymine- adenine (T-A) change in an non-coding intron region of a gene, resulting in alternative splicing and translation of immunogenic or cryptic peptide sequences. In some embodiments, the deamination of the target C base results in a C- G basepair to thymine- adenine (T-A) change in the start (Met) codon of the open reading frame of a gene, resulting in the translation of an alternative open reading frame comprising immunogenic or cryptic peptide sequences.

[0069] Other aspects of the present disclosure provide compositions comprising: (i) a fusion protein comprising (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a cytosine deaminase domain; and (ii) a guide nucleotide sequence targeting the fusion protein of (i) to a polynucleotide encoding a tumor specific antigen.

[0070] Yet other aspects of the present disclosure provide compositions comprising a polynucleotide encoding a fusion protein and a guide nucleotide sequence, wherein the fusion protein comprises (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a cytosine deaminase domain, and wherein the guide nucleotide sequence targets the fusion protein to a polynucleotide encoding a tumor specific antigen.

[0071] Yet other aspects of the present disclosure provide cancer vaccines comprising: (i) a fusion protein comprising (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a cytosine deaminase domain; and (ii) a guide nucleotide sequence targeting the fusion protein of (i) to a polynucleotide encoding a tumor specific antigen.

[0072] Further provided herein are cancer vaccine comprising a polynucleotide encoding a fusion protein and a guide nucleotide sequence, wherein the fusion protein comprises (a) a guide nucleotide sequence-programmable DNA-binding protein domain; and (b) a cytosine deaminase domain, and wherein the guide nucleotide sequence targets the fusion protein to a polynucleotide encoding a tumor specific antigen.

[0073] Kits comprising the cancer vaccines described herein are also provided.

[0074] The details of certain embodiments of the disclosure are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the disclosure will be apparent from the Definitions, Examples, Figures, and Claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the disclosure and together with the description, serve to explain the principles of the disclosure.

[0076] Figure 1 shows strategies to engineer heteroclitic and cryptic epitopes using genome base editing.

[0077] Figure 2A shows strategies to introduce immunogenic heteroclitic epitopes by editing conservative anchor residues to match the binding preference of the main HLA allele supertypes. The example shows a base-editing reaction that turns an alanine residue at anchor position 9 of a weakly immunogenic peptide epitope into a preferred valine residue for binding HLA-A2.

[0078] Figure 2B shows anchor-residue binding preference and population coverage of the main HLA allele supertypes (MHC-I pathway). The peptides in Figure 2B are as follows: HLA Al, A2, A3, and A24 (SEQ ID NOs: 878-881) and HLA B7, B27, B44, B58, and B62 (SEQ ID NOs: 882-886).

[0079] Figure 3 shows a proposed mechanism for anti-cancer vaccination by

heteroclitic/cryptic epitopes introduced by genome base-editing reactions programmed by guide-RNAs. The edited tumor cells produce heteroclitic and cryptic epitopes in cancer- specific genes, which chemotactically attract immature dendritic cells (DCs) (la), inducing DC maturation (lb). Edited tumor cells produce apoptotic bodies (2a) that are taken up by DCs (2b), contributing to maturation of DCs (lb) and B cells (2b'). Mature DCs activate CD4+ T lymphocytes (lc) and CD8+ T lymphocytes (lc'). Activated CD4+ T lymphocytes further stimulate B-lymphocyte activation (Id) and provide IL-2 for CD8+ T lymphocytes (Id'). B lymphocytes produce TAA-specific antibodies to cell-surface proteins that result in antibody-dependent cell-mediated cytotoxicity or complement-mediated tumor cell death (le). Activated CD8+ T lymphocytes then kill tumor cells via recognition of MHC class I molecules in association with TAA epitopes (le').

[0080] Figure 4 shows comparison of cancer lineages that display high frequency of mutagenesis, which may harbor non- synonymous hitchhiker mutations and "neo-epitopes".

DEFINITIONS

[0081] As used herein and in the claims, the singular forms "a," "an," and "the" include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to "an agent" includes a single agent and a plurality of such agents. [0082] An "immunogenic peptide" or "antigenic peptide" is a peptide or epitope that can be recognized by the immune system and elicit an immune response. Immunogenic peptides or antigenic peptide may comprise a motif such that the peptide will bind an MHC molecule and induce a T cell response, or can be recognized by the B cell receptor on the B cell to induce antibody production. These terms are used interchangeably herein.

[0083] An "immunogenic epitope" or "antigenic epitope" refers to a part of an antigen is recognized by the immune system, e.g., by antibodies, B cells, or T cells. In some

embodiments, the epitope is the specific piece of the antigen to which an antibody binds. Although epitopes are usually non-self proteins, sequences derived from the host can, in some instances, be recognized.

[0084] "Immune response" is how your body recognizes and defends itself against bacteria, viruses, and substances that appear foreign and harmful to the body. In its general form, the immune response begins with the sensitization of helper (TH, CD4+) and cytotoxic (CD8+) T cell subsets through their interaction with antigen presenting cells (APC) that express major histocompatibility (MHC)-class I or class II molecules associated with antigenic fragments (i.e., specific amino acid sequences derived from the antigen which bind to MHC I and/or MHC II for presentation on the cell surface). The sensitized or primed CD4+ T cells produce lymphokines that participate in the activation of B cells as well as various T cell subsets. The sensitized CD8+ T cells increase in numbers in response to lymphokines and are capable of destroying any cells that express the specific antigenic fragments associated with matching MHC-encoded class I molecules. Thus, in the course of a cancerous tumor, CTL eradicate cells expressing cancer associated or cancer specific antigens, thereby limiting the

progression of tumor spread and disease development.

[0085] The "adaptive immune system," also known as the acquired immune system, is a subsystem of the overall immune system that is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogen growth. The adaptive immune system is one of the two main immunity strategies found in vertebrates (the other being the innate immune system). Adaptive immunity creates immunological memory after an initial response to a specific pathogen, and leads to an enhanced response to subsequent encounters with that pathogen. This process of acquired immunity is the basis of vaccination. Like the innate system, the adaptive system includes both humoral immunity components and cell-mediated immunity components.

[0086] Unlike the innate immune system, the adaptive immune system is highly specific to a particular pathogen or antigen. Adaptive immunity can also provide long-lasting protection. The adaptive system response destroys invading pathogens and any toxic molecules they produce. In accordance with the present disclosure, the adaptive immune system response destroys tumor or cancer cells. Sometimes the adaptive system is unable to distinguish harmful from harmless foreign molecules. The cells that carry out the adaptive immune response are white blood cells known as lymphocytes. Two main broad classes— antibody responses and cell mediated immune response— are also carried by two different

lymphocytes (B cells and T cells). In antibody responses, B cells are activated to secrete antibodies, which are proteins also known as immunoglobulins. Antibodies travel through the bloodstream and bind to the foreign antigen causing it to inactivate, which does not allow the antigen to bind to the host.

[0087] In adaptive immunity, pathogen-specific receptors are "acquired" during the lifetime of the organism (whereas in innate immunity pathogen- specific receptors are already encoded in the germline). The acquired response is called "adaptive" because it prepares the body's immune system for future challenges (though it can actually also be maladaptive when it results in autoimmunity).

[0088] The immune system is highly adaptable because of somatic hypermutation (a process of accelerated somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Since the gene rearrangement leads to an irreversible change in the DNA of each cell, all progeny (offspring) of that cell inherit genes that encode the same receptor specificity, including the memory B cells and memory T cells that are the keys to long-lived specific immunity.

[0089] A " T cell" or "T lymphocyte" is a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor on the cell surface. They are called T cells because they mature in the thymus from thymocytes. The several subsets of T cells each have a distinct function. The majority of human T cells rearrange their alpha and beta chains on the cell receptor and are termed alpha beta T cells (αβ T cells) and are part of the adaptive immune system. Specialized gamma delta T cells, (a small minority of T cells in the human body, more frequent in ruminants), have invariant T cell receptors with limited diversity, that can effectively present antigens to other T cells and are considered to be part of the innate immune system. Effector T cell broadly includes various T cell types that actively respond to a stimulus, such as co- stimulation. This includes helper, killer, regulatory, and potentially other T cell types. One skilled in the art is familiar with different types of T cells and their respective roles in adaptive immune response.

[0090] A "human leukocyte antigen (HLA) system" is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. These cell-surface proteins are responsible for the regulation of the immune system in humans. The HLA gene complex resides on a 3 Mbp stretch within chromosome 6p21. HLA genes are highly polymorphic, which means that they have many different alleles, allowing them to fine-tune the adaptive immune system. The proteins encoded by certain genes are also known as antigens, as a result of their historic discovery as factors in organ transplants. Different classes have different functions:

[0091] HLAs encoding major histocompatibility complex (MHC) class I MHC class I (A, B, and C) molecules, which present peptides from inside the cell. "Major histocompatibility complex (MHC) class I" or "MHC class I" molecules are found on the cell surface of all nucleated cells in the body. Their function is to display peptide fragments of antigens from within the cell to cytotoxic T cells; this will trigger an immediate response from the immune system against a particular non-self antigen displayed with the help of an MHC class I protein. Because MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called cytosolic or endogenous pathway.

[0092] Class I MHC molecules bind peptides generated mainly from degradation of cytosolic proteins by the proteasome. The MHC I peptide complex is then inserted via endoplasmic reticulum into the external plasma membrane of the cell. The epitope peptide is bound on extracellular parts of the class I MHC molecule. Thus, the function of the class I MHC is to display intracellular proteins to cytotoxic T cells (CTLs). However, class I MHC can also present peptides generated from exogenous proteins, in a process known as cross- presentation.

[0093] A normal cell will display peptides from normal cellular protein turnover on its class I MHC, and CTLs will not be activated in response to them due to central and peripheral tolerance mechanisms. When a cell expresses foreign proteins, such as after viral infection, a fraction of the class I MHC will display these peptides on the cell surface. Consequently, CTLs specific for the MHC:peptide complex will recognize and kill presenting cells.

Alternatively, class I MHC itself can serve as an inhibitory ligand for natural killer cells (NKs). Reduction in the normal levels of surface class I MHC, a mechanism employed by some viruses during immune evasion or in certain tumors, will activate NK cell killing. Antigens or antigenic epitopes presented by MHC class II molecules are recognized by cytotoxic T cells.

[0094] HLAs encoding MHC class II (DP, DM, DOA, DOB, DQ, and DR) molecules, which present antigens from outside of the cell to T-lymphocytes. "Major histocompatibility complex class Π" or "MHC class Π" molecules are a family of molecules normally found only on antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells. The antigens presented by class II peptides are usually derived from extracellular proteins (not cytosolic as in class I); hence, the MHC class II-dependent pathway of antigen presentation is called the endocytic or exogenous pathway. Loading of a MHC class II molecule occurs by phagocytosis;

extracellular proteins are endocytosed, digested in lysosomes, and the resulting epitopic peptide fragments are loaded onto MHC class II molecules prior to their migration to the cell surface. Antigens or antigenic epitopes presented by MHC class II molecules are recognized by T helper cells and stimulate the multiplication of T-helper cells, which in turn stimulate antibody-producing B-cells to produce antibodies to that specific antigen. Self-antigens are suppressed by regulatory T cells.

[0095] An "antigen-presenting cell (APC)" is a cell that displays antigen complexed with major histocompatibility complexes (MHCs) on their surfaces; this process is known as antigen presentation. T cells may recognize these complexes using their T cell receptors (TCRs). These cells process antigens and present them to T-cells. Antigen-presenting cells fall into two categories: professional and non-professional. Those that express MHC class II molecules along with co- stimulatory molecules and pattern recognition receptors are often called professional antigen-presenting cells. The non-professional APCs express MHC class I molecules.

[0096] Professional APCs specialize in presenting antigen to T cells. They are very efficient at internalizing antigens, either by phagocytosis (macrophages and dendritic cells) or by receptor-mediated endocytosis (B cells), processing the antigen into peptide fragments and then displaying those peptides, bound to a class II MHC molecule, on their membrane. [1] The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. An additional co-stimulatory signal is then produced by the antigen-presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules and MHC class II are defining features of professional APCs.

[0097] Almost all cell types can serve as a non-professional APC. They are found in a variety of tissue types. Professional antigen-presenting cells, including dendritic cells, mononuclear phagocytes, thymic epithelial cells, and B cells, present foreign antigens to helper T cells, while other cell types can present antigens originating inside the cell to cytotoxic T cells. In addition to the MHC family of proteins, antigen presentation relies on other specialized signaling molecules on the surfaces of both APCs and T cells.

[0098] A "B lymphocyte" or "B cell" is a type of white blood cell of the lymphocyte subtype. B cells function in the humoral immunity component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigen (they are also classified as professional antigen-presenting cells (APCs)) and secrete cytokines. In mammals, B cells mature in the bone marrow, which is at the core of most bones. B cells express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind a specific antigen, against which it will initiate an antibody response.

[0099] "Cancer immunotherapy" refers to a type of cancer treatment designed to boost the body's natural defenses to fight the cancer. It uses substances either made by the body or in a laboratory to improve or restore immune system function.

[0100] "Tumor specific antigen (TSA)" or "tumor associated antigen (TAA)" refers to a protein that is specifically expressed or upregulated in cells of the respective tumor, as compared to non-cancerous cells of the same origin. A tumor specific antigen, or epitopes derived therefrom, can be recognized by the immune system to induce a immune response. Herein, the terms "tumor associated antigen" and "tumor specific antigen" are used interchangeably. The tumor specific antigen may be from all protein classes, e.g., enzymes, receptors, transcription factors, etc.

[0101] A "heteroclitic epitope" or "heteroclitic analog" refers to an altered version of an endogenous peptide sequence {i.e., an analog) engineered to elicit potent immune reactions. Heteroclitic epitopes have increased stimulatory capacity or potency for a specific T cell, as measured by increased responses to a given dose, or by a requirement of lesser amounts to achieve the same response and therefore provide benefit as vaccine components since these epitopes induce T cell responses stronger than those induced by the native epitope.

[0102] A "self-antigen" refers to an antigen that originates from within the body. The immune system usually does not react to self-antigens under normal homeostatic conditions. Epitopes from self-antigens {i.e., self-epitopes) are found in high concentration on the surface of Antigen-presenting cells (APCs) in association with its major histocompatibility complex (MHC) are known as dominant epitopes. These are stimulants of negative selection mechanisms to remove potentially self-destructing autoreactive T cells. Their "self" antigens are displayed to a developing T-cell and signal those "self-reactive" T-cells to die via programmed cell death (apoptosis) and thereby deletion from the T cell repertoire, preventing autoimmunity.

[0103] A "cryptic epitope" refers to an epitope derived from a self-antigen that does not necessarily undergo antigen processing/presentation and are 'hidden' from immune recognition. Cryptic epitopes usually appear in very low concentration on APC and do not delete auto-reactive T cells. Cryptic epitopes are not presented for recognition by T cells unless they are produced in unusually large concentrations or unless they are freed from the configuration of their native antigen. Cryptic epitopes derived from tumor- specific antigens may be used to break the tolerance of T cells to the tumor and induce potent immune response against the tumor. Such principles have been described in Pardoll, et al., PNAS, Vol. 96, pp. 5340-5342 (1999), the entire contents of which are incorporated herein by reference.

[0104] A "neoepitope" refers to an antigenic epitope generated via random somatic mutations occurring in tumor cells. Neoepitopes are usually derived from individually specific tumor antigens or unique antigens and is thus specific to the lineage of tumor cells it is derived from. Neoepitopes are regarded in the art to be responsible for the immunogenicity of tumors ((Srivastava et al., 1993, Duan et al., 2009; van der Bruggen et al., 2013), and mathematic modeling has predicted the existence of tens to hundreds of neoepitopes in individual human tumors (Srivastava 2009). The recent revolution in high-throughput DNA sequencing and accompanying bioinformatics approaches has finally made it possible to actually identify the individually specific neoepitopes in individual cancers.

[0105] "Cancer vaccine," as used herein, refers to a composition that induces tumor- specific immunoresponse against a tumor or a tumor- specific antigen. Such immunoresponse is effective in inhibiting tumor growth and/or preventing reoccurrence of tumor.

[0106] An "intron" refers to any nucleotide sequence within a gene that is removed by RNA splicing during maturation of the final RNA product. The term intron refers to both the DNA sequence within a gene and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final mature RNA after RNA splicing are exons. Introns are found in the genes of most organisms and many viruses, and can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA). When proteins are generated from intron-containing genes, RNA splicing takes place as part of the RNA processing pathway that follows transcription and precedes translation.

[0107] An "exon" refers to any part of a gene that will become a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature messenger RNA.

[0108] "RNA splicing" refers to the processing of a newly synthesized messenger RNA transcript (also referred to as a primary mRNA transcript). After splicing, introns are removed and exons are joined together (ligated) for form mature mRNA molecule containing a complete open reading frame that is decoded and translated into a protein. For nuclear- encoded genes, splicing takes place within the nucleus either co-transcriptionally or immediately after transcription. The molecular mechanism of RNA splicing has been extensively described, e.g., in Pagani et al, Nature Reviews Genetics 5, 389-396, 2004;

Clancy et ah, Nature Education 1 (1): 31, 2011; Cheng et ah, Molecular Genetics and Genomics 286 (5-6): 395-410, 2014; Taggart et al, Nature Structural & Molecular Biology 19 (7): 719-2, 2012, the contents of each of which are incorporated herein by reference. One skilled in the art is familiar with the mechanism of RNA splicing.

[0109] "Alternative splicing" refers to a regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. Consequently, the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions . Notably, alternative splicing allows the human genome to direct the synthesis of many more proteins than would be expected from its 20,000 protein-coding genes.

Alternative splicing is sometimes also termed differential splicing. Alternative splicing occurs as a normal phenomenon in eukaryotes, where it greatly increases the biodiversity of proteins that can be encoded by the genome; in humans, -95% of multi-exonic genes are alternatively spliced. There are numerous modes of alternative splicing observed, of which the most common is exon skipping. In this mode, a particular exon may be included in mRNAs under some conditions or in particular tissues, and omitted from the mRNA in others. Abnormal variations in splicing are also implicated in disease; a large proportion of human genetic disorders result from splicing variants. Abnormal splicing variants are also thought to contribute to the development of cancer, and splicing factor genes are frequently mutated in different types of cancer. The regulation of alternative splicing is also described in the art, e.g., in Douglas et al., Annual Review of Biochemistry 72 (1): 291-336, 2003; Pan et al, Nature Genetics 40 (12): 1413-1415, 2008; Martin et al, Nature Reviews 6 (5): 386-398, 2005; Skotheim et ah, The international journal of biochemistry & cell biology 39 (7-8): 1432-49, 2007, the entire contents of each of which is incorporated herein by reference.

[0110] A "coding frame" or "open reading frame" refers to a streich of codons that encodes a polypeptide. Since DNA is interpreted in groups of three nucleotides (codons), a DNA strand has three distinct reading frames. The double helix of a DNA molecule has two anti- parallel strands so, with the two strands having three reading frames each, there are six possible frame translations. A functional protein may be produced when translation proceeds in the correct coding frame. An insertion or a deletion of one or two bases in the open reading frame causes a shift in the coding frame that is also referred to as a "frameshift mutation." A frameshift mutation typical results in premature translation termination and/or truncated or non-functional protein.

[0111] The term "proteome" refers to the entire set of proteins expressed by a genome, cell, tissue, or organism at a certain time. More specifically, it is the set of expressed proteins in a given type of cell or organism, at a given time, under certain conditions. The term is a blend of proteins and genome. "Proteome-wide" refers to each and every protein in the proteome without any bias.

[0112] The term "genome" refers to the genetic material of a cell or organism. It typically includes DNA (or RNA in the case of RNA viruses). The genome includes both the genes, the coding regions, the noncoding DNA, and the genomes of the mitochondria and chloroplasts. A genome does not typically include genetic material that is artificially introduced into a cell or organism, e.g., a plasmid that is transformed into a bacteria is not a part of the bacterial genome.

[0113] A "programmable DNA-binding protein," as used herein, refers to DNA binding proteins that can be programmed to navigate to any desired target nucleotide sequence within the genome. To program the DNA-binding protein to bind a desired nucleotide sequence, the DNA binding protein may be modified to change its binding specificity, e.g., zinc finger nuclease (ZFN) or transcription activator-like effector proteins (TALE). ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA- cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. Transcription activator- like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, Methods of programming ZFNs and TALEs are familiar to one skilled in the art. For example, such methods are described in Maeder, et al, Mol. Cell 31 (2): 294-301, 2008; Carroll et al, Genetics Society of America, 188 (4): 773-782, 2011; Miller et al, Nature Biotechnology 25 (7): 778-785, 2007; Christian et al, Genetics 186 (2): 757-61, 2008; Li et al, Nucleic Acids Res 39 (1): 359-372, 2010; and Moscou et al, Science 326 (5959): 1501, 2009, the entire contents of each of which are incorporated herein by reference.

[0114] A "guide nucleotide sequence-programmable DNA-binding protein," as used herein, refers to a protein, a polypeptide, or a domain that is able to bind DNA, and the binding to its target DNA sequence is mediated by a guide nucleotide sequence. Thus, it is appreciated that the guide nucleotide sequence-programmable DNA-binding protein binds to a guide nucleotide sequence. The "guide nucleotide" may be a RNA molecule or a DNA molecule {e.g., a single- stranded DNA or ssDNA molecule) that is complementary to the target sequence and can guide the DNA binding protein to the target sequence. In some

embodiments, the guide nucleotide sequence is an oligonucleotide sequence. As such, a guide nucleotide sequence-programmable DNA-binding protein may be a RNA-programmable DNA-binding protein {e.g., a Cas9 protein), or an ssDNA-programmable DNA-binding protein {e.g., an Argonaute protein). "Programmable" means the DNA-binding protein may be programmed to bind any DNA sequence that the guide nucleotide targets.

[0115] In some embodiments, the guide nucleotide sequence exists as a single nucleotide molecule and comprises comprise two domains: (1) a domain that shares homology to a target nucleic acid {e.g., and directs binding of a guide nucleotide sequence-programmable DNA-binding protein to the target); and (2) a domain that binds a guide nucleotide sequence- programmable DNA-binding protein. In some embodiments, the guide nucleotide is a guide RNA (gRNA). In some embodiments, domain (2) of the gRNA corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al, Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs {e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S.S.N. 61/874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases And Uses Thereof," and U.S. Provisional Patent Application, U.S.S.N. 61/874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire contents of each are hereby incorporated by reference in their entirety. [0116] Because the guide nucleotide sequence hybridizes to target DNA sequence, the guide nucleotide sequence-programmable DNA-binding proteins are able to be targeted, in principle, to any sequence specified by the guide nucleotide sequence. Methods of using guide nucleotide sequence-programmable DNA-binding protein, such as Cas9, for site- specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Science 339, 819-823 (2013); Mali, P. et al. Science 339, 823-826 (2013); Hwang, W.Y. et al. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. eLife 2, e00471 (2013);

Dicarlo, J.E. et al. Nucleic acids research (2013); Jiang, W. et al. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0117] It is to be understood that any DNA binding domain that is programmable by a guide nucleotide sequence may be used in accordance with the present disclosure. For example, in some embodiments, the guide nucleotide sequence-programmable DNA binding protein may be a Cas9 protein, or a variant thereof. One skilled in the art would understand that the present disclosure is not limited to the use of Cas9 as the guide nucleotide sequence- programmable DNA binding protein, but that other DNA binding proteins that adopt similar mechanism of target sequence binding may also be used.

[0118] As used herein, the term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 protein, a fragment, or a variant thereof. A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3- aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5'

exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs ("sgRNA", or simply "gNRA") can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek et al., Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. [0119] Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., Ferretti et al, Proc. Natl. Acad. Sci. 98:4658-4663(2001); Deltcheva E. et al, Nature 471:602-607(2011); and Jinek et al, Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus . Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski et ah, (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2, SEQ ID NO: 4 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 1 (amino acid).

ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCG GTGATCAC

TGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCA CAGTATCA

AAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTC GTCTCAAA

CGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAG ATTTTTTCA

AATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTG GTGGAAGAA

GACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTAT CATGAGAAA

TATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGAT TTGCGCTTA

ATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGA GATTTAAATC

CTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAAT TATTTGAAG

AAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGA GTAAATCAA

GACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTG GGAATCTCA

TTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAG ATGCTAAATT

ACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGG AGATCAATA

TGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATAT CCTAAGAGTA

AATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAA CATCATCAA

GACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAA ATCTTTTTT

GATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAA TTTTATAA

ATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACT AAATCGTGA

AGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCA CTTGGGTGA

GCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCG TGAGAAGAT

TGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAA TAGTCGTTTT

GCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTT GTCGATAA

AGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCC AAATGAAAA

AGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGAC AAAGGTCAA

ATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGC CATTGTTG ATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCA AAAAA

ATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCA TTAGGTACC

TACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAAT GAAGATATC

TTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAA AGACTTAAA

ACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTAT ACTGGTTGG

GGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACA ATATTAGAT

TTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT AGTTTGACAT

TTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAAC ATATTGCA

AATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTT GATGAATTG

GTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAA AATCAGAC

AACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTAT CAAAGAA

TTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAA AAGCTCTAT

CTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAAT CGTTTAAGT

GATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGAC AATAAGGTC

TTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTA GTCAAAAA

GATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTT TGATAATTT

AACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACG CCAATTGG

TTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATA CTAAATACG

ATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAG TTTCTGACT

TCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCC ATGATGCGT

ATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGG AGTTTGTCT

ATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAA TAGGCAAA

GCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATT ACACTTGCA

AATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATT GTCTGGGA

TAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATAT TGTCAAGAA

AACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTC GGACAAGC

TTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAA CGGTAGCTT

ATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCG TTAAAGAG

TTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTT TTAGAAGCT

AAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTT TTTGAGTTA

GAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAG CTGGCTCT

GCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGG TAGTCCAGA

AGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGAT TATTGAGC

AAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTC TTAGTGCAT

ATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTAT TTACGTTG

ACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAA CGATATACG

TCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTAT GAAACACGC

ATTGATTTGAGTCAGCTAGGAGGTGACTGA (SEQ ID NO: 4) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEV AYHEKYPTIYHLR

KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDA

KAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDN

LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEK

YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHL

GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGA

SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFK

TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLT

LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRN

FMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVK VMGRHKPENIVI

EMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLON GRDMYVDQEL

DINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRO LLNAKLITQRK

FDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSD

FRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEOEIGKA

TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM POVNIVKKTEVO

1GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIME

RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLA

SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENI

IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO:

1) (single underline: HNH domain; double underline: RuvC domain)

[0120] In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus aureus. S. aureus Cas9 wild type (SEQ ID NO: 6)

MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRR RHRIQRVKK

LLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQI

SRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQ SFIDTYIDLLE

TRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLN NLVITRDENE

KLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYH DIKDITARKEIIEN

AELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIA

IFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPND IIIELAREKNSKDAQK

MINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDL LNNPFNYEVDHIIP

RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGR ISKTKKEYLLEER

DINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRK WKFKKERNKGY

KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFI TPHQIKHIKDFK

DYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDP

QTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHL DITDDYPNSR

NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISN QAEFIASFYNN DLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKK YSTDILGNLYE VKSKKHPQIIKKG

[0121] In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus thermophilus.

Streptococcus thermophilus wild type CRISPR3 Cas9 (St3Cas9)

MTKPYSIGLDIGTNSVGWAVITDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAE GRRLKRTA

RRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEE KVYHDEFPTIYHL

RKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAI FESDLSLENSKQ

LEEIVKDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCFNLDEKASL HFSKESYDEDLETL

LGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYNEHKEDLALL KEYIRNISLKTYN

EVFKDDTKNGYAGYIDGKTNQEDFYVYLKNLLAEFEGADYFLEKIDREDFLRKQRTF DNGSIPYQIHLQ

EMRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKIT PWNFEDVIDKESS

AEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSKQ KKDIVRLYFKDK

RKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEA IIEEIIHTLTIFEDRE

MIKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDG ISNRNFMQLIHDD

ALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRK PESIVVEMARENQ

YTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNG KDMYTGDDLDI

DRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDFPSLEVVKKRKTFWYQLL KSKLISQRKFDNL

TKAERGGLLPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRAVRTVKIIT LKSTLVSQFRKD

FELYKVREINDFHHAHDAYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERKSAT EKVYFYSNIMNI

FKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQN HGLDRGKPKGL

FNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKKKITN VLEFQGISILDRI

NYRKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGN QIFLSQKFVKLLYH

AKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQSWQNH SIDELCSSFIGPT

GSERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRID LAKLGEG (SEQ ID

NO: 7)

Streptococcus thermophilus CRISPR1 Cas9 wild type (StlCas9)

MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLTRRKK HRRVRLNRL

FEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASD DGNSSIGDYAQIVK

ENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRIL QTQQEFNPQITDE

FINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR AAKASYTAQEFNL

LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGY RIDKSGKAEIHT

FEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQV DELVQFRKANS

SIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLT EEIYNPVVAKSVR

QAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQ YNGKAELPHSV

FHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLA NKVLVYATANQE

KGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIE RNLVDTRYASR VVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLN LWKKQKN

TLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDS KFNRKISDATIYAT

RQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIE PILENYPNKQI

NEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNN KVVLQSVSPWR

ADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTL YKNDLLLVKDT

ETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGK SNISIYKVRTD

VLGNQHIIKNEGDKPKLDF (SEQ ID NO: 8)

[0122] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein is a Cas9 from archaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, the Cas9 domain is CasX or CasY, which have been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038/cr.2017.21, which is incorporated herein by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp) and are within the scope of this disclosure.

[0123] In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally- occurring CasX or CasY protein. In some embodiments, the Cas9 domain is a naturally- occurring CasX or CasY protein. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 336-337 or 3000. In some embodiments, the Cas9 domain comprises an amino acid sequence of any one SEQ ID NOs: 336-337 or 3000. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0124] In some embodiments, wild-type Cas9 refers to CasX from Sulfolobus islandicus (strain REY15A). MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAE

RRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQV

KECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKA

KVS EGD Y VG VN VFTPTRGILYS LIQN VNGIVPGIKPET AFGLWIARKV VS S VTNPN VS

VVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRER YIV

LANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

(SEQ ID NO: 338)

[0125] In some embodiments, wild-type Cas9 refers to CasX from Sulfolobus islandicus (strain REY15A).

MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAE RRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQV KECEE VS APS FVKPEF YEFGRS PGM VERTRRVKLE VEPH YLIIA A AG W VLTRLGKAK VS EGD YVGVN VFTPTRGILYS LIQN VNGIVPGIKPET AFGLWIARKV VS S VTNPN VS V VRIYTIS D A VGQNPTTINGGFS IDLTKLLEKRYLLS ERLE AIARN ALS IS S NMRERYIVL ANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG

(SEQ ID NO: 339)

[0126] In some embodiments, wild-type Cas9 refers to CasY from a Parcubacteria group bacterium.

CasY (ncbi.nlm.nih.gov/protein/APG80656.1)

>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]

MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAIND DYVGLYGLSNFD

DLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKG SHLYDELQID

KVIKFLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGA SLGERQKKLFR

DFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNE GSLEMWEYIGIG

NSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELEKRLRILAALT IKLREPKFDNHW

GGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVV SSLLESIEKIVP

DDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKR KKKSDAEDEKETI

DFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSL KNSFFDTDFDK

DFFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSA AIDKNRVRLPSTE

NIAKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDIS ALDFVENGTVKD

FMKTRDGNLVLEGRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELL YIPHEFQSAKITT

PKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELTRTGQGIDGG VAENALRLEKSP

VKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLI DEKKVKTRWN

YDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITG DSAKILDQNFISDPQ

LKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYEL EVSRFEEGKQKIK

KVYATLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQV DETITTQELIG

TVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPF CRANADADIQASQ

TIALLRYVKEEKKVEDYFERFRKLKNIKVLGQMKKI (SEQ ID NO: 3000) [0127] In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs:

NC 016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1);

Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1); Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria, meningitidis (NCBI Ref: YP_002342100.1) or to a Cas9 from any of the organisms listed in Example 1 (SEQ ID NOs: 11-260).

[0128] To be used as in the fusion protein of the present disclosure as the guide nucleotide sequence-programmable DNA binding protein domain, a Cas9 protein needs to be nuclease inactive. A nuclease-inactive Cas9 protein may interchangeably be referred to as a "dCas9" protein (for nuclease-"dead" Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et ah, Science. 337:816-821(2012); Qi et al, (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et ah, Science. 337:816-821(2012); Qi et al, Cell. 28;152(5): 1173-83 (2013)). dCas9 (D10A and H840A)

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEV AYHEKYPTIYHLR

KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDA

KAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDN

LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEK

YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHL

GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGA

SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFK

TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLT

LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRN

FMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVK VMGRHKPENIVI EMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLONGRD MYVDQEL

DINRLSDYDVDAIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRO LLNAKLITQRK

FDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSD

FRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEOEIGKA

TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM POVNIVKKTEVO

1GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIME

RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLA

SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENI

IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO:

2) (single underline: HNH domain; double underline: RuvC domain)

[0129] The dCas9 of the present disclosure encompasses completely inactive Cas9 or partially inactive Cas9. For example, the dCas9 may have one of the two nuclease domain inactivated, while the other nuclease domain remains active. Such a partially active Cas9 may also be referred to as a Cas9 nickase, due to its ability to cleave one strand of the targeted DNA sequence. The Cas9 nickase suitable for use in accordance with the present disclosure has an active HNH domain and an inactive RuvC domain and is able to cleave only the strand of the target DNA that is bound by the sgRNA. The Cas9 nickase of the present disclosure may comprise mutations that inactivate the RuvC domain, e.g. , a D10A mutation. It is to be understood that any mutation that inactivates the RuvC domain may be included in a Cas9 nickase, e.g., insertion, deletion, or single or multiple amino acid substitution in the RuvC domain. In a Cas9 nickase described herein, while the RuvC domain is inactivated, the HNH domain remains activate. Thus, while the Cas9 nickase may comprise mutations other than those that inactivate the RuvC domain (e.g., D10A), those mutations do not affect the activity of the HNH domain. In a non-limiting Cas9 nickase example, the histidine at position 840 remains unchanged. The sequence of an exemplary Cas9 nickase suitable for the present disclosure is provided below.

Cas9 Nickase (D10A)

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEV AYHEKYPTIYHLR

KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDA

KAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDN

LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEK

YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHL

GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGA SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK AIVDLLFK

TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLT

LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRN

FMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVK VMGRHKPENIVI

EMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLON GRDMYVDQEL

DINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRO LLNAKLITQRK

FDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSD

FRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEOEIGKA

TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM POVNIVKKTEVO

1GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIME

RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLA

SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENI

IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO:

3) (single underline: HNH domain; double underline: RuvC domain)

S. aureus Cas9 Nickase (D10A)

MKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRR RHRIQRVKK

LLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQI

SRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQ SFIDTYIDLLE

TRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLN NLVITRDENE

KLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYH DIKDITARKEIIEN

AELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIA

IFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPND IIIELAREKNSKDAQK

MINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDL LNNPFNYEVDHIIP

RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGR ISKTKKEYLLEER

DINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRK WKFKKERNKGY

KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFI TPHQIKHIKDFK

DYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDP

QTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHL DITDDYPNSR

NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISN QAEFIASFYNN

DLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQS IKKYSTDILGNLYE

VKSKKHPQiiKKG (SEQ ID NO: 5)

[0130] It is appreciated that when the term "dCas9" or "nuclease-inactive Cas9" is used herein, it refers to Cas9 variants that are inactive in both HNH and RuvC domains as well as Cas9 nickases. For example, the dCas9 used in the present disclosure may include the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the dCas9 may comprise other mutations that inactivate RuvC or HNH domain. Additional suitable mutations that inactivate Cas9 will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D839A and/or N863A (See, e.g., Prashant et al, Nature Biotechnology. 2013; 31(9): 833- 838, the entire contents of which are incorporated herein by reference), or K603R {See, e.g., Chavez et ah, Nature Methods 12, 326-328, 2015, the entire contents of which is

incorporated herein by reference). The term Cas9, dCas9, or Cas9 variant also encompasses Cas9, dCas9, or Cas9 variant from any organism. Also appreciated is that dCas9, Cas9 nickase, or other appropriate Cas9 variants from any organisms may be used in accordance with the present disclosure.

[0131] A "deaminase" refers to an enzyme that catalyzes the removal of an amine group from a molecule, or deamination. In some embodiments, the deaminase is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase is a cytosine deaminase, catalyzing the hydrolytic deamination of cytosine to uracil {e.g., in RNA) or thymine {e.g., in DNA). In some embodiments, the deaminase is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.

[0132] A "cytosine deaminase" refers to an enzyme that catalyzes the chemical reaction "cytosine + H ₂ 0 ->uracil + NH ₃ " or "5-methyl-cytosine + H20 -> thymine + NH3." As it may be apparent from the reaction formula, such chemical reactions result in a C to U/T nucleobase change. In the context of a gene, such nucleotide change, or mutation, may in turn lead to an amino acid residue change in the protein, which may affect the protein function, e.g., loss-of-function or gain-of-function.

[0133] One exemplary suitable class of cytosine deaminases is the apolipoprotein B mRNA-editing complex (APOBEC) family of cytosine deaminases encompassing eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner. The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-1 strain via the deamination of cytosines in reverse-transcribed viral ssDNA. These cytosine deaminases all require a Zn ²⁺ -coordinating motif (His-X-Glu-X ₂₃ _ ₂ 6- Pro-Cys-X ₂ _ ₄ -Cys; SEQ ID NO: 820) and bound water molecule for catalytic activity. The Glu residue acts to activate the water molecule to a zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own particular "hotspot", ranging from WRC (W is A or T, R is A or G) for hAID, to TTC for hAPOBEC3F. A recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure comprised of a five-stranded β-sheet core flanked by six a-helices, which is believed to be conserved across the entire family. The active center loops have been shown to be responsible for both ssDNA binding and in determining "hotspot" identity.

Overexpression of these enzymes has been linked to genomic instability and cancer, thus highlighting the importance of sequence-specific targeting. Another suitable cytosine deaminase is the activation-induced cytidine deaminase (AID), which is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription- dependent, strand-biased fashion.

[0134] Herein, a "nucleobase editor" refers to to a protein that edits a nucleotide base.

"Edit" refers to the conversion of one nucleotide base to another. For example, the

nucleobase may target C bases in a nucleic acid sequence and convert the C to T base. In some embodiments, the C to T editing is carried out by a deaminase, e.g., a cytosine deaminase. Other types of base conversions are also contemplated. In some embodiments, the nucleobase editor comprises a DNA binding domain that directs it to a target sequence.

[0135] As such, a base editor may be a cytosine deaminase-dCas9 fusion protein. In some embodiments, the base editor may be a deaminase-dCas9-UGI fusion protein. In some embodiments, the base editor may be a APOBECl-dCas9-UGI fusion protein. In some embodiments, the base editor may be APOBECl-Cas9 nickase-UGI fusion protein. In some embodiments, the base editor may be APOBECl-dCpfl-UGI fusion protein. In some embodiments, the base editor may be APOBECl-dNgAgo-UGI fusion protein. In some embodiments, the base editor may be a pmCDAl-Cas9 nickase-UGI fusion protein. In some embodiments, the base editor may be a human APOBEC3G-Cas9 nickase UGI fusion protein. In some embodiments, the base editor may comprise a second UGI domain. Non- limiting exemplary sequences of the nucleobase editors described herein are provided in Example 1, SEQ ID NOs: 293-302, 1071, and 1084. Such nucleobase editors and methods of using them for genome editing have been described in the art, e.g., in US Patent 9,068,179, US Patent Application Publications US 2015/0166980, US 2015/0166981, US 2015/0166982, US20150166984, and US20150165054, and US Provisional Applications, U.S.S.N.

62/245,828, filed October 23, 2015; 62/279,346, filed January 15, 2016; 62/311,763, filed March 22, 2016; 62/322178, filed April 13, 2016, 62/357,352, filed June 30, 2016,

62,370,700, filed August 3, 2016; 62/398,490, filed September 22, 2016; 62/408686, filed October 14, 2016; PCT Application PCT/US2016/058344, filed October 22, 2016, US Patent Application 15/311,852, filed October 22, 2016; Komor et al. (2017) Improved Base Excision Repair Inhibition and Bateriophage Mu Gam Protein Yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 3: eaao4774; and in Komor et ah, Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), the entire contents of each of which are incorporated herein by reference.

[0136] The term "target site" or "target sequence" refers to a sequence within a nucleic acid molecule (e.g. , a DNA molecule) that is deaminated by the fusion protein provided herein. In some embodiments, the target sequence is a polynucleotide (e.g., a DNA), wherein the polynucleotide comprises a coding strand and a complementary strand. The meaning of a "coding strand" and "complementary strand" is the common meaning of the terms in the art. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human. The term "target codon" refers to the amino acid codon that is edited by the base editor and converted to a different codon via deamination of C base. In some embodiments, the target codon is edited in the coding strand. In some embodiments, the target codon is edited in the complementary strand.

[0137] The term "linker," as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain). Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g. , a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 2- 100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90- 100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0138] The term "mutation," as used herein, refers to a substitution of a residue within a sequence, e.g. , a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[0139] The terms "nucleic acid" and "nucleic acid molecule," as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and

"polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g. , a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3 ' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g. , methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages).

[0140] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term "fusion protein" as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0141] The term "subject," as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

[0142] The term "recombinant" as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0143] The immune system is critical in preventing the outgrowth of cancers, and

"immuno surveillance" exists to provide immunological resistance against cancer

development (e.g., as described in Old et al., Annu Rev Med 1964; 15: 167-186; Burnet et ah, Prog Exp Tumor Res 1970; 13: 1-27; and Graziano et ah, Cancer Treat Res 2005; 123: 89- 111, each of which is incorporated herein by reference).

[0144] Despite the presence of immunosurveillance, cancers can develop in apparently immunocompetent animals and humans, due to the ability of cancer cells to evade

immunosurveillance (e.g. , as described in Hanahan et ah, Cell 2000; 100: 57-70 and Zitvogel et ah, Nat Rev Immunol 2006; 6: 715-727, each of which in incorporated herein by reference). The evasion of cancer cells from immunosurveillance occurs via various well- characterized mechanisms, including induction of T-cell tolerance by autochthonous tumors (e.g., as described in Willimsky et ah, Nature 2005; 437: 141-146, incorporated herein by reference), cancer immunoediting (e.g., as described in Dunn et ah, Nat Immunol 2002; 3: 991-998, incorporated herein by reference), and development of an immune suppressive cancer microenvironment (e.g., as described in Zou et ah, Nat Rev Cancer 2005; 5: 263-274, incorporated herein by reference). Therapeutic cancer vaccines or adoptive immunotherapy are being developed and tested as potential approaches to strengthen the immune responses after tumor arise in order to slow their progression and prevent their recurrence.

Immunotherapeutic approaches, e.g., cancer vaccines have been described but are only partially successful (e.g., as described in Finn et ah, Nat Rev Immunol 2003; 3: 630-641, incorporated herein by reference).

[0145] Described herein are systems, methods, compositions, and kits for producing immunogenic peptides derived from tumor specific antigens (e.g., heteroclitic epitopes or cryptic epitopes) that may be used as cancer vaccines in vivo or ex vivo. Targeted mutations are introduced into tumor- specific antigens using a gene editing agent, e.g., a nucleobase editor comprising a programmable DNA binding domain (e.g., a catalytically-inactive Cas9 or Cas9 nickase) fused to a cytosine deaminase, to generate altered versions of peptides arising from the tumor- specific antigens (heteroclitic epitopes) or peptides arising from normally untranslated regions of the tumor- specific antigen genes (cryptic peptides). The heteroclitic peptides or cryptic peptides may be generated in vivo in a subject (e.g., a subject who has cancer) and presented to the adaptive immune system via the MHC class I or MHC class II pathway, which in turn induces a strong adaptive immune response, e.g., T cell response and B cell response. Such an adaptive immune response is antigen specific and is effective in reducing tumor growth and preventing metastasis.

[0146] The advantage of the cancer vaccines of the present disclosure is that the vaccine (e.g., antigenic peptides derived from tumor- specific antigens) is generated from the genome and the proteome of the malignant cells in vivo and is highly personalized. The cancer vaccines described herein are also highly cancer- specific and do not induce unwanted immune response against "self," since the immunogenic epitopes are derived from tumor- specific antigens. Further, the adaptive immune response induced by the cancer vaccine described herein confer "memory" to the immune system, promoting the immune system to efficiently recognize "neoepitopes" generated due to the highly mutagenic nature of the cancer genome, thus preventing metastasis and facilitate remission. To enhance the efficacy of the cancer vaccines described herein, combination therapies using an immune checkpoint inhibitor in conjunction with the cancer vaccine is also contemplated, aiming to enhance the tumor antigen specific immune response.

[0147] The methods of producing endogenous cancer vaccines in vivo are enabled by the targeted nucleobase editing technology described herein. Such base editing technology is described in the art, e.g., in US Patent 9,068,179, US Patent Application Publications US 2015/0166980, US 2015/0166981, US 2015/0166982, US20150166984, and

US20150165054, and US Provisional Applications, U.S.S.N. 62/245,828, filed October 23, 2015; 62/279,346, filed January 15, 2016; 62/311,763, filed March 22, 2016; 62/322178, filed April 13, 2016, 62/357,352, filed June 30, 2016, 62,370,700, filed August 3, 2016; 62/398,490, filed September 22, 2016; 62/408686, filed October 14, 2016; PCT Application PCT/US2016/058344, filed October 22, 2016, US Patent Application 15/311,852, filed October 22, 2016; and in Komor et ah, Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), the entire contents of each of which are incorporated herein by reference.

Immunogenic Peptides or Epitopes

[0148] Some aspects of the present disclosure provide immunogenic peptides or epitopes derived from tumor- specific antigens and how these peptides or epitopes elicit tumor- specific immune response. A large number of proteins that specifically express in tumor cells or are upregulated in tumor cells have been identified (Hassane et al., Holland-Frei Cancer

Medicine. 6th edition). The known tumor specific antigens are classified into different classes.

[0149] a) Cancer-testis antigens: The first TAAs ever identified that can be recognized by T cells belong to this class, which was originally called cancer-testis (CT) antigens because of the expression of its members in histologically different human tumors and, among normal tissues, only in spermatocytes/spermatogonia of testis and, occasionally, in placenta. Since the cells of testis do not express class I and II HLA molecules, these antigens cannot be recognized by T cells in normal tissues and can therefore be considered as immunologically tumor- specific. Well-known examples of CT antigens are the MAGE family members or NY- ESO-1.

[0150] b) Differentiation antigens: These TAAs are shared between tumors and normal tissue from which the tumor arose; most are found in melanomas and normal melanocytes. Many of these melanocyte lineage-related proteins are involved in the biosynthesis of melanin and are therefore not tumor specific but nevertheless are widely used for cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan- A/MART- 1 for melanoma, and PSA for prostate cancer.

[0151] c) Overexpressed TAAs: Genes encoding widely expressed TSAs have been detected in histologically different types of tumors as well as in many normal tissues, generally with lower expression levels. It is possible that many of the epitopes processed and potentially presented by normal tissues are below the threshold level for T-cell recognition, while their overexpression in tumor cells can trigger an anticancer response by breaking previously established tolerance. Examples of this class of TAAs are Her-2/neu, Survivin, Telomerase and WT1. [0152] d) Tumor specific antigens: These unique TAAs arise from mutations of normal genes (such as β-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and/or progression. Tumor specific antigens are generally able to induce strong immune responses without bearing the risk for autoimmune reactions against normal tissues. On the other hand, these TAAs are in most cases only relevant to the exact tumor on which they were identified and are usually not shared between many individual tumors.

[0153] e) TAAs arising from abnormal post-translational modifications: Such TSAs may arise from proteins which are neither specific nor overexpressed in tumors but nevertheless become tumor associated by posttranslational processes primarily active in tumors. Examples for this class arise from altered glycosylation patterns leading to novel epitopes in tumors (e.g., MUC1).

[0154] f) Qncoviral proteins: These TSAs are viral proteins that may play a critical role in the oncogenic process, and because they are foreign (not of human origin), they can evoke a T-cell response. Examples of such proteins are the human papilloma type 16 virus proteins, E6 and E7, which are expressed in cervical carcinoma.

[0155] TAAs are a starting point for the development of a tumor vaccine. The methods for identifying and characterizing the TAAs are based on the use of cytoxic T lymphocytes (CTL) that can be isolated from patients or healthy subjects, or they are based on the generation of differential transcription profiles or differential peptide expression patterns between tumors and normal tissues.

[0156] In some embodiments, the tumor- specific antigen is expressed in a broad range of different types of cancers. In some embodiments, the tumor- specific antigen is expressed only in one or a few types of cancers. The anti-cancer immune response described herein is antigen-specific. As such, an immune response induced by a tumor- specific antigen is specific to cancer types where the said antigen is expressed. Non-limiting, exemplary tumor- specific antigens that may be edited to generate immunogenic epitopes are provided in Tables 1-3. It is appreciated that the examples are for illustration purpose only, and the methods described herein may be applied to any tumor- specific antigen.

[0157] In some embodiments, the immunogenic peptide or epitope is a portion of the tumor- specific antigen. For example, the immunogenic peptide or epitope may be a portion of the tumor- specific antigen that is 5-40 amino acids long. In some embodiments, the immunogenic peptide or epitope is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long. In some embodiments, the immunogenic peptide or epitope comprises modifications, e.g., amino acid substitutions (also termed "heteroclitic epitopes"), as compared to the native sequence in the tumor specific antigen. In some embodiments, the immunogenic peptide or epitope comprises more than one amino acid substitutions (e.g. , 2, 3, 4, 5, or more) compared to the native sequence of the tumor- specific antigen it is derived from. In some embodiments, a heteroclitic peptide or epitope may be at least 60%, at least 70%, at least 80%, at least 90%, at least 98%, or at least 99% identical to the native sequence that it is derived from. In some embodiments, a heteroclitic peptide or epitope is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the native sequence that it is derived from.

[0158] In some embodiments, a heteroclitic peptide or epitope is more immunogenic than a peptide of its native sequence. For example, a heteroclitic epitope may be at least 30% more immunogenic (i.e., induces a stronger immune response) than its corresponding native peptide. In some embodiments, a heteroclitic epitope may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or more immunogenic than its corresponding native peptide.

[0159] In some embodiments, the immunogenic peptide or epitope is a cryptic peptide or epitope, e.g. , generated from translation of a non-coding region of the tumor specific antigen gene or translation of a different reading frame of a coding region of the tumor specific antigen. A cryptic peptide or epitope may be more immunogenic (i.e., induces a stronger immune response) than any native peptide derived from the tumor associated antigen. For example, a cryptic peptide or epitope may be at least 30% more immunogenic than any native peptide derived from the tumor associated antigen. In some embodiments, a cryptic peptide or epitope may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80- fold, at least 90-fold, at least 100-fold, or more immunogenic than any native peptide derived from the tumor associated antigen. One skilled in the art is familiar with how to assess the immune response induced by an antigen, e.g., measuring antibody titers. [0160] Tumor specific antigens from which antigenic epitopes (e.g., heteroclitic epitopes and cryptic epitopes) may be derived are provided in Tables 1-3. (HLA - human leukocyte antigen type)

Table 1. Tumor associated antigen - Differentiation

Table 2. Tumor specific antigen - Tumor specific

Table 3. Overexpressed tumor specific antigen

[0161] The identification of heteroclitic peptides are described in the art. For example, in previous studies (Selby, et ah, J. Immunol., 162(2):669 (1999), Skipper, et ah, J. Exp. Med. 183:527 (1996), the entire contents of each of which are incorporated herein by reference), heteroclitic epitopes were fortuitously identified by eluting naturally occurring mutant peptides from melanoma cells, or by systematically screening a large number of epitopes consisting of substitutions at almost every position in the epitope (Zaremba, et ah, Cancer Research, 57:4570 (1997), Loftus, et al, Cancer Research 58:2433 (1998), Blake, et al, J. Exp. Med. 18: 121 (1996), the entire contents of each of which are incorporated herein by reference). Alternatively, heteroclitic epitopes were identified by screening random combinatorial peptide libraries which also has required the arduous synthesis and screening of large numbers of peptides (Pinilla, et ah, Current Opinion in Immunology 11: 193-202 (1999), the entire contents of each of which are incorporated herein by reference). Genetic approaches, such as screening of DNA expression libraries, have provided another method for generating CTL epitopes and analogs (Boon, et al., Annu. Rev. Immunol. 12:337-65 (1994), Gavin, et al, Eur. J. Immunol. 24(9):2124-33 (1994), the entire contents of each of which are incorporated herein by reference).

Generating Cancer Vaccine in Tumor Cells

[0162] Some aspects of the present disclosure provide systems, compositions, and methods of editing genes encoding tumor specific antigens in vivo (e.g., in tumor cells in a subject) or ex vivo (e.g., in isolated tumor cells) to introduce mutations in the genes encoding tumor specific antigens. In some embodiments, such mutations lead to the production of heteroclitic peptides that are more immunogenic than native peptides of the tumor-specific antigen. In some embodiments, such mutations lead to the translation of a non-coding region of the tumor specific antigen, which results in cryptic peptides that are more immunogenic than any native peptides from the tumor specific antigen.

[0163] The gene editing methods described herein, rely on nucleobase editors as described in ., in US Patent 9,068,179, US Patent Application Publications US 2015/0166980, US 2015/0166981, US 2015/0166982, US20150166984, and US20150165054, and US

Provisional Applications, U.S.S.N. 62/245,828, filed October 23, 2015; 62/279,346, filed January 15, 2016; 62/311,763, filed March 22, 2016; 62/322178, filed April 13, 2016, 62/357,352, filed June 30, 2016, 62,370,700, filed August 3, 2016; 62/398,490, filed

September 22, 2016; 62/408686, filed October 14, 2016; PCT Application

PCT/US2016/058344, filed October 22, 2016, US Patent Appliation 15/311,852, filed October 22, 2016; and in Komor et ah, Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), the entire contents of each of which are incorporated herein by reference. [0164] The nucleobase editors are highly efficient at precisely editing a target base in any of the tumor associated antigen genes described herein, and a DNA double stand break is not necessary for the gene editing, thus reducing genome instability and preventing possible oncogenic modifications that may be caused by other genome editing methods. The nucleobase editors described herein may be programmed to target and modify a single base. In some embodiments, the target base is a cytosine (C) base and may be converted to a thymine (T) base via deamination by the nucleobase editor.

[0165] To edit the polynucleotide encoding a tumor associated antigen, the polynucleotide is contacted with a nucleobase editor as described herein. In some embodiments, the tumor- associated antigen encoding polynucleotide is contacted with a nucleobase editor and a guide nucleotide sequence, wherein the guide nucleotide sequence targets the nucleobase editor the target base (e.g. , a C base) in the tumor-associated antigen encoding polynucleotide.

[0166] In some embodiments, the tumor-associated antigen encoding polynucleotide is the tumor-associated antigen gene locus in the genomic DNA of a cell (e.g., a tumor cell). In some embodiments, the tumor cell is a cultured cell. In some embodiments, the tumor cell is in vivo. In some embodiments, the tumor cell is ex vivo. In some embodiments, the tumor cell is from a mammal. In some embodiments, the mammal is a human. In some

embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat.

[0167] In some embodiments, the tumor-associated antigen encoding polynucleotide may be a DNA molecule comprising a coding strand and a complementary strand, e.g., the tumor- associated antigen gene locus in the genome of a tumor cell. In some embodiments, the tumor-associated antigen encoding polynucleotide may also include coding regions (e.g., exons) and non-coding regions (e.g., introns or splicing sites). In some embodiments, the target base (e.g. , a C base) is located in the coding region (e.g. , an exon) of the tumor- associated antigen encoding polynucleotide. As such, the conversion of a base in the coding region may result in an amino acid change in the tumor-associated antigen protein sequence, i.e. , a mutation. Tumor associated antigens comprising the desired mutation(s), once degraded (e.g. , via any of the protein degradation pathways, such as degradation by the proteasome) results in immunogenic heteroclitic epitopes.

[0168] In some embodiments, the target base is located in a non-coding region of the tumor-associated antigen gene, e.g. , in an intron or a splice site. In some embodiments, a target base is located in a splice site, and the editing of such target base causes alternative splicing of the tumor-associated antigen mRNA. In some embodiments, the alternative splicing leads to translation of a non-coding region of the tumor-associated antigen gene, generating cryptic epitopes. The immunogenic epitopes (e.g. , heteroclitic epitopes or cryptic epitopes) may be presented by the tumor cell, or a professional antigen presenting cell, and be recognized by the immune system, thus eliciting a tumor- specific immune response (e.g. , T- cell response or B-cell response).

[0169] To edit a tumor-associated antigen gene, the tumor-associated antigen gene (a polynucleotide molecule) may be contacted with the nucleobase editor, wherein the nucleobase editor binds to its target sequence and edits the desired base. For example, the nucleobase editor may be expressed in a cell where editing is desired (e.g., a tumor), allowing editing of the tumor-associated antigen gene by the nucleobase editor. In some embodiments, the binding of the nucleobase editor to its target sequence in the tumor-associated antigen gene is mediated by a guide nucleotide sequence, e.g., a nucleotide molecule comprising a nucleotide sequence that is complementary to one of the strands of the target sequence in the tumor-associated antigen gene. Thus, by designing the guide nucleotide sequence, the nucleobase editor may be programmed to edit any target base in any tumor associated antigen gene. In some embodiments, the guide nucleotide sequence is co-expressed with the nucleobase editor in a tumor cell where editing is desired. In some embodiments, a nucleobase editor/gRNA complex is delivered to the cell where editing is desired (e.g., a tumor cell).

[0170] Provided herein are non-limiting, exemplary heteroclitic epitopes and cryptic epitopes that may be produced via base editing and strategies for making them.

Codon Change

[0171] Using the nucleobase editors described herein, several amino acid codons may be converted to a different codon via deamination of a target base within the codon. For example, in some embodiments, a cytosine (C) base is converted to a thymine (T) base via deamination by a nucleobase editor comprising a cytosine deaminase domain (e.g. ,

APOBEC 1 or AID). It is worth noting that during a C to T change via deamination (e.g., by a cytosine deaminase such as APOBEC 1 or AID), the cytosine is first converted to a uridine (U), leading to a G:U mismatch. The G:U mismatch is then converted by DNA repair and replication pathways to T:A pair, thus introducing the thymine at the position of the original cytosine. As such, deamination of a C base results in a C-G base pair being replaced by a T-A base pair. [0172] As is familiar to one skilled in the art, conversion of a base in an amino acid codon may lead to a change of the amino acid the codon encodes. Cytosine deaminases are capable of converting a cytosine (C) base to a thymine (T) base via deamination. Thus, it is envisioned that, for amino acid codons containing a C base, the C base may be directly converted to T. For example, codon for leucine (CTC) may be changed to a TTC

(phenylalanine) codon via the deamination of the first C on the coding strand. For amino acid codons that contain a guanine (G) base, a C base is present on the complementary strand; and the G base may be converted to an adenosine (A) via the deamination of the C on the complementary strand. For example, an ATG (Met/M) codon may be converted to a ATA (Ile/I) codon via the deamination of the third C on the complementary strand. In some embodiments, two C to T changes are required to convert a codon to a different codon. Non- limiting examples of possible mutations that may be made in any tumor associated antigens by the nucleobase editors of the present disclosure are summarized in Table 4.

[0173] Such amino acid substitutions introduced via base editing generate heteroclitic epitopes. Non-limiting examples of heteroclitic epitopes that may be generated from tumor associated antigens by nucleobase editors are summarized in Table 5. [0174] In some embodiments, to bind to its target sequence and edit the desired base, the nucleobase editor depends on its guide nucleotide sequence (e.g., a guide RNA). In some embodiments, the guide nucleotide sequence is a gRNA sequence. A gRNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to fusion proteins disclosed herein. In some embodiments, the guide RNA comprises the structure 5 '-[guide sequence] - guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaag uggcaccgagucggugcuu uuu-3 ' (SEQ ID NO: 336), wherein the guide sequence comprises a sequence that is complementary to the target sequence. Other suitable tracrRNA framework sequences are provided in Table 11. The guide sequence is typically about 20 nucleotides long. In certain embodiments, the guide sequence may be 15-25 nucleotides long. In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 (e.g., within 50, 45, 40, 35, 30, 35, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10) nucleotides upstream or downstream of the target nucleotide to be edited.

[0175] Guide sequences that may be used to target the nucleobase editor to its target sequence to induce specific mutations in tumor associated antigen genes are provided in Table 5. It is to be understood that the mutations and guide sequences presented herein are for illustration purpose only and are not meant to be limiting.

Table 5. Heteroclitic epitopes

Cryptic Epitopes

[0176] Some aspects of the present disclosure provide strategies for generating cryptic epitopes in tumor cells. In some embodiments, such strategies involve alterations of splicing sites in a tumor associated antigen gene. Altered splicing site may lead to altered splicing of an mRNA that encodes a tumor associated antigen. One outcome of altered splicing is the translation of an otherwise non-coding region of the gene, leading to otherwise "hidden peptides," i.e., cryptic epitopes. The splicing site typically comprises an intron donor site, a Lariat branch point, and an intron acceptor site. The mechanism of splicing are familiar to those skilled in the art. The intron donor site has a consensus sequence of GGGTRAGT, and the C bases paired with the G bases in the intron donor site consensus sequence may be targeted by a nucleobase editor, thereby altering the intron donor site. The Lariat branch point also has a consensus sequence, e.g., YTRAC, wherein Y is a pyrimidine, and R is a purine. The C base in the Lariat branch point consensus sequence may be targeted by the nucleobase editors described herein, leading to skipping of the following exon. The intron acceptor site has a consensus sequence of YNCAGG, wherein Y is a pyrimidine, and N is any nucleotide. The C base of the consensus sequence of the intron acceptor site, and the C base paired with the G bases in the consensus sequence of the intron acceptor site may be targeted by the nucleobase editors described herein, thereby altering the intron acceptor site, in turn leading to skipping of an exon. General strategies of altering the splicing sites are described in Table 6.

[0177] Non-limiting, exemplary cryptic epitopes that may be produced using the base editing methods described herein are provided in Table 7.

[0178] In some embodiments, the nucleobase editor may be used to introduce a premature stop codon (a stop codon that occurs upstream of the normal stop codon) into a tumor specific antigen gene (e.g. , TAA, TAG, and TGA). In some embodiments, introduction of a premature stop codon destabilizes the tumor specific antigen. In some embodiments, destabilization of the tumor specific antigen leads to enhanced presentation of immunogenic epitopes (e.g. , heteroclitic epitopes or cryptic epitopes).

[0179] Premature stop codons are introduced by changing one or more bases in a target codon that encodes a target residue. For example, nucleobase editors including a cytosine deaminase domain are capable of converting a cytosine (C) base to a thymine (T) base via deamination. Thus, it is envisioned that, for amino acid codons containing a C base, the C base may be converted to T. For example, a CAG (Gln/Q) codon may be changed to a TAG (amber) codon via the deamination of the first C on the coding strand. For sense codons that contain a guanine (G) base, a C base is present on the complementary strand; and the G base may be converted to an adenosine (A) via the deamination of the C on the complementary strand. For example, a TGG (Trp/W) codon may be converted to a TAG (amber) codon via the deamination of the second C on the complementary strand. In some embodiments, two C to T changes are required to convert a codon to a nonsense codon. For example, a CGG (R) codon is converted to a TAG (amber) codon via the deamination of the first C on the coding strand and the deamination of the second C on the complementary strand.

[0180] In some embodiments, the target residue is located in a flexible loop region of the tumor specific antigen. In some embodiments, tandem premature stop codons are introduced. Non-limiting examples of codons that may be changed to stop codons via base editing are provided in Table 8.

[0181] In some embodiments, cryptic epitopes are generated by shifting the coding frame of a tumor specific antigen gene. In some embodiments, the coding frame is shifted by changing a start codon (ATG) to a sense codon that cannot be used as a start codon. As such, translation will start at the next start codon in the coding region, and the coding frame may be shifted. In some embodiments, a normal sense codon may be edited to generate a start codon to allow translation to start at the newly generated start codon, which may also lead to shifting of the coding frame. Alterations of start codons and the resulting shift in the coding frame generate peptides that would not otherwise be generated from the tumor specific antigen gene (i.e., cryptic epitopes). Non-limiting, exemplary start codon alterations that may be achieved by the nucleobase editors described herein are provided in Table 9.

[0182] The tumor associated antigens listed in Table 5 and Table 6 and their respective gene sequences and protein sequences are known in the art. The amino acid sequence of the listed tumor specific antigens are listed in Table 10. Table 10. Amino acid sequences of human tumor specific antigens

(SPAG11A)

BCR/ABL MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKAS IRRLEQEVNQE 778

RFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPQPAPADG

fusion protein ADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPASVAALRS

NFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI SSLGSQAM

isoform X3 QMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAELNPRFLKDNL

IDANGGSRPPWPPLEYQPYQSIYVGGMMEGEGKGPLLRSQSTSEQEKRLTWP RRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQSSRVSPSPTTYRMFR DKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVWSEATIVGVRKTGQIWPN DGEGAFHGDADGSFGTPPGYGCAADRAEEQRRHQDGLPYIDDSPSSSPHLSS KGRGSRDALVSGALESTKASELDLEKGLEMRKWVLSGILASEETYLSHLEAL LLPMKPLKAAATTSQPVLTSQQIETIFFKVPELYEIHKEFYDGLFPRVQQWS HQQRVGDLFQKLASQLGVYRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNS LEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRS I SLRYEGRVYH YRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTV YGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDT MEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYI ITEFMTYGNLLDYLRE CNRQEVNAWLLYMATQI SSAMEYLEKKNFIHRDLAARNCLVGENHLVKVAD FGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFS IKSDVWAFGVLLWEIA TYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPS FAEIHQAFETMFQESS I SDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRR AAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDER LLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDI SNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTL TSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGR QFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEV FKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAGKGSALGTPAAAE PVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPP PPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGE GLKKPVLPATPKPQSAKPSGTPI SPAPVPSTLPSASSALAGDQPSSTAFIPL I STRVSLRKTRQPPERIASGAITKGWLDSTEALCLAI SRNSEQMASHSAVL EAGKNLYTFCVSYVDS IQQMRNKFAFREAINKLENNLRELQICPATAGSGPA ATQDFSKLLSSVKEISDIVQR

Carcinoembry MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVL 779

LLVHNLPQHLFGYSWYKGERVDGNRQI IGYVIGTQQATPGPAYSGREI IYPN

onic antigen- ASLLIQNI IQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPS I SSNNSKP

VEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRN

related cell DTASYKCETQNPVSARRSDSVILNVLYGPDAPTI SPLNTSYRSGENLNLSCH

AASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTT

adhesion VTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLP

VSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPD

molecule 5 DPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNIT (CEACAM5) EKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPS I SSNNSKPVEDKDAVA

FTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCG IQNSVSANRSDPVTLDVLYGPDTPI I SPPDSSYLSGANLNLSCHSASNPSPQ YSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNS IVKS ITVSA SGTSPGLSAGATVGIMIGVLVGVALI

Melanoma MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGE 780

VPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESE

antigen, FQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSWGNWQYFFPVIFSKAFS

SLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLI IVLAI

family A, 3 IAREGDCAPEEKIWEELSVLEVFEGREDS ILGDPKKLLTQHFVQENYLEYRQ

VPGSDPACYEFLWGPRALVETSYVKVLHHMVKI SGGPHI SYPPLHEWVLREG

variant EE

(MAGE- A3)

Mucin- 1 MTPGTQSPFFLLLLLTVLTWTGSGHASSTPGGEKETSATQRSSVPSSTEKN 781

AVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVT

(MUC1) RPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH

GVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP

[0183] In some embodiments, cancer vaccines containing immunogenic peptides from tumor specific antigens (e.g., heteroclitic epitopes and cryptic epitopes) are generated in vivo (e.g., in tumor cells in a subject) or ex vivo (e.g., in tumor cells isolated from a subject). In some embodiments, the tumor cells are treated with the nucleobase editors to generate the immunogenic peptides and are irradiated and administered to the subject as whole-cell cancer vaccines.

[0184] To edit the genes encoding the tumor associated antigens, the nucleobase editor and/or the guide nucleotide sequence is introduced into the cell (e.g., a tumor cell) where the editing occurs. In some embodiments, nucleic acid molecules (e.g., expression vectors) encoding the nucleobase editors and/or the guide nucleotide sequences are delivered into the cell, resulting in co-expression of nucleobase editors and/or the guide nucleotide sequences in the cell. The nucleic acid molecules encoding the nucleobase editors and/or the guide nucleotide sequences may be delivered into the cell using any known methods in the art, e.g., transfection (e.g., transfection mediated by cationic liposomes), transduction (e.g., via viral infection) and electroporation. In some embodiments, an isolated nucleobase editor/gRNA complex is delivered. Methods of delivering an isolated protein to a cell is familiar to those skilled in the art. For example, the isolated nucleobase editor in complex with a gRNA be associated with a supercharged, cell-penetrating protein or peptide, which facilitates its entry into a cell (e.g., as described in PCT Application Publication WO2010129023 and US Patent Application Publication US20150071906, incorporated herein by reference). In some embodiments, the isolated nucleobase editor in complex with a gRNA may be delivered by a cationic transfection reagent, e.g., the Lipofectamine CRISPRMAX Cas9 Transfection Reagent from Thermofisher Scientific. In some embodiments, the nucleobase editor and the gRNA may be delivered separately. Other suitable delivery methods may also be used, e.g., AAV mediated gene transfer. Strategies for delivery a Cas9-based genome editing agent (e.g., the nucleobase editor described herein) using AAV have been described, e.g., in Zetsche et al., Nature Biotechnology 33, 139-142 (2015), incorporated herein by reference.

[0185] In some embodiments, once generated, the immunogenic peptide is displayed on the surface of the tumor cell via the MHC class I antigen presentation pathway. In some embodiments, the immunogenic peptide is displayed on the surface of an antigen presenting cell (APC) via the MHC class II antigen presentation pathway. In some embodiments, the APC is selected from the group consisting of: tumor cells, dendritic cells, mononuclear phagocytes, thymic epithelial cells, and B cells. In some embodiments, the immunogenic peptide elicits an adaptive immune response against the tumor- specific antigen where the peptide is derived from. In some embodiments, the immunogenic peptide elicits an adaptive immune response against the tumor. In some embodiments, the adaptive immune response comprises promoting the maturation of dendritic cells, activation of CD4+T lymphocytes, (T helper cells) activation of CD8+ T lymphocytes (cytotoxic T cells), activation and maturation of B lymphocytes, and/or production of tumor antigen-specific antibodies.

[0186] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surfaces. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THl, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate different types of immune responses. Signaling from the APC directs T cells into particular subtypes. [0187] Cytotoxic T cells (e.g., TC cells, CTLs, T-killer cells, killer T cells) destroy virus- infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surfaces. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0188] Most cytotoxic T cells express T-cell receptors (TCRs) that can recognize a specific antigen. Antigens inside a cell are bound to class I MHC molecules, and brought to the surface of the cell by the class I MHC molecule, where they can be recognized by the T cell. If the TCR is specific for that antigen, it binds to the complex of the class I MHC molecule and the antigen, and the T cell destroys the cell, e.g., via inducing apoptosis. In order for the TCR to bind to the class I MHC molecule, the former must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule. Therefore, these T cells are called CD8+ T cells.

[0189] Natural killer T cells (NKT cells - not to be confused with natural killer cells of the innate immune system) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major

histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic/cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with microorganisms, e.g., bacteria or virus.

[0190] Memory T cells are a subset of antigen- specific T cells that persist long-term after an initial T cell response. They quickly expand to large numbers of effector T cells upon re- exposure to their cognate antigen, thus providing the immune system with "memory" against past antigens. The cancer vaccine described herein provides the immune system with

"memory" against the tumor specific antigen, thereby eliciting strong immune response against newly emerged cancer cells or metastasized cancer cells.

[0191] Regulatory T cells (suppressor T cells) are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus. Suppressor T cells along with Helper T cells can collectively be called Regulatory T cells due to their regulatory functions. [0192] B cell activation occurs in the secondary lymphoid organs (SLOs), such as the spleen and lymph nodes. After B cells mature in the bone marrow, they migrate through the blood to SLOs, which receive a constant supply of antigen through circulating lymph. At the SLO, B cell activation begins when the B cell binds to an antigen via its BCR. The antigen can either be free-floating or presented by APCs such as macrophages or dendritic cells (DCs), and include proteins, glycoproteins, polysaccharides, whole virus particles, and whole bacterial cells. Some subtypes of B cell preferentially undergo T cell-dependent activation while other subtypes of cells preferentially undergo T cell-independent activation.

[0193] Antigens that activate B cells with the help of T-cell are known as T cell-dependent (TD) antigens and include foreign proteins. They are named as such because they are unable to induce a humoral response in organisms that lack T cells. B cell response to these antigens takes multiple days, though antibodies generated have a higher affinity and are more functionally versatile than those generated from T cell-independent activation.

[0194] Once a BCR binds a TD antigen, the antigen is taken up into the B cell through receptor-mediated endocytosis, degraded, and presented to T cells as peptide pieces in complex with MHC-II molecules on the cell membrane. T helper (TH) cells, typically follicular T helper (TFH) cells, that were activated with the same antigen recognize and bind these MHC-II-peptide complexes through their T cell receptor (TCR). Following TCR-MHC- II-peptide binding, T cells express the surface protein CD40L as well as cytokines such as IL- 4 and IL-21. CD40L serves as a necessary co- stimulatory factor for B cell activation by binding the B cell surface receptor CD40, which promotes B cell proliferation,

immunoglobulin class switching, and somatic hypermutation as well as sustains T cell growth and differentiation. T cell-derived cytokines bound by B cell cytokine receptors also promote B cell proliferation, immunoglobulin class switching, and somatic hypermutation as well as guide differentiation. After B cells receive these signals, they are considered activated.

[0195] Activated B cells participate in a two-step differentiation process that yields both short-lived plasmablasts for immediate protection and long-lived plasma cells and memory B cells for persistent protection. The first step, known as the extrafollicular response, occurs outside of lymphoid follicles but still in the SLO. During this step activated B cells proliferate, may undergo immunoglobulin class switching, and differentiate into plasmablasts that produce early, weak antibodies mostly of class IgM. The second step consists of activated B cells entering a lymphoid follicle and forming a germinal center (GC), which is a specialized microenvironment where B cells undergo extensive proliferation,

immunoglobulin class switching, and affinity maturation directed by somatic hypermutation. These processes are facilitated by TFH cells within the GC and generate both high-affinity memory B cells and long-lived plasma cells. Resultant plasma cells secrete large amounts of antibody and either stay within the SLO or, more preferentially, migrate to bone marrow.

[0196] Antigens that activate B cells without T cell help are known as T cell-independent (TI) antigens and include foreign polysaccharides and unmethylated CpG DNA. They are named as such because they are able to induce a humoral response in organisms that lack T cells. B cell response to these antigens is rapid, though antibodies generated tend to have lower affinity and are less functionally versatile than those generated from T cell-dependent activation.

[0197] As with TD antigens, B cells activated by TI antigens need additional signals to complete activation, but instead of receiving them from T cells, they are provided either by recognition and binding of a common microbial constituent to toll-like receptors (TLRs) or by extensive crosslinking of BCRs to repeated epitopes on a bacterial cell. B cells activated by TI antigens go on to proliferate outside of lymphoid follicles but still in SLOs (GCs do not form), possibly undergo immunoglobulin class switching, and differentiate into short-lived plasmablasts that produce early, weak antibodies mostly of class IgM, but also some populations of long-lived plasma cells.

[0198] Memory B cell activation begins with the detection and binding of their target antigen, which is shared by their parent B cell. Some memory B cells can be activated without T cell help, such as certain virus -specific memory B cells, but others need T cell help. Upon antigen binding, the memory B cell takes up the antigen through receptor- mediated endocytosis, degrades it, and presents it to T cells as peptide pieces in complex with MHC-II molecules on the cell membrane. Memory T helper (TH) cells, typically memory follicular T helper (TFH) cells, that were derived from T cells activated with the same antigen recognize and bind these MHC-II-peptide complexes through their TCR. Following TCR- MHC-II-peptide binding and the relay of other signals from the memory TFH cell, the memory B cell is activated and differentiates either into plasmablasts and plasma cells via an extrafollicular response or enter a germinal center reaction where they generate plasma cells and more memory B cells.

[0199] In some embodiments, the adaptive immune response results in the killing tumor cells, reducing tumor burden, reducing tumor size, and/or preventing metastasis. In some embodiments, the adaptive immune response is active against neo-epitopes associated with spontaneous somatic mutations. In some embodiments, the neo-epitope is specific to the lineage of tumor cells. [0200] In some embodiments, the adaptive immune response elicited by the heteroclitic or cryptic epitopes is cross-reactive with the native tumor- specific antigen. In some

embodiments, the adaptive immune response elicited by the heteroclitic or cryptic epitopes is cross-reactive with neoepitopes arising from spontaneous mutations occurring in the tumor specific antigen.

[0201] There are advantages associated with using heteroclitic epitopes in clinical applications. For example, heteroclitic epitopes have the ability to break/overcome tolerance by reversing a state of T cell anergy, activating non-tolerized cross -reactive clones of T cells, or by mediating "immune deviation," i.e., the type of CTL produced, such as Thl or Th2. Recent studies indicate that heteroclitic epitopes are immunogenic (Zaremba, et al, Cancer Research, 57:4570 (1997); Rivoltoni, et al, Cancer Research, 59:301 (1999); Selby, et al, The Journal of Immunology 162(2):669 (1999), the entire contents of each of which are incorporated herein by reference) in that they are capable of inducing CTLs that recognize endogenously processed epitopes. This is confirmed by studies in different immunological systems (Zugel, et al., J. Immunol., 161: 1705 (1998), Wang, et ah, J. Exp. Med., 190:983 (1999), Men, et ah, J. Immunol., 162:3566, (1999), the entire contents of each of which are incorporated herein by reference). For example, studies by Zugel et al. have shown that T cell tolerance to an immunodominant T cell epitope in adult mice can be overcome by

immunization with heteroclitic cross-reactive peptide analogs of that peptide.

[0202] In some embodiments, heteroclitic epitopes or cryptic epitopes modulate cytokine production from T cells (Pfeiffer, et al., J. Exp. Med., 181: 1569 (1995), Tao, et al., J.

Immunol, 158:4237 (1997), Salazar, et al, Int. J. Cancer 85(6):829-38 (2000), Nicholson, et al, Int. Immunol. 12(2):205-13 (2000), the entire contents of each of which are incorporated herein by reference). The immune deviation induced by such analogs has implications in several disease states, where generation of a specific subset of Th cell responses correlate with tumor regression (Zitvogel, et al, J. Exp. Med., 183:87 (1996), Celluzzi, et al, J. Exp. Med. 183:283 (1996), the entire contents of each of which are incorporated herein by reference) or affected the clinical outcome of autoimmune or infectious disease (Romagnani, et al., Annu. Rev. Immunol, 12:227-57 (1994), the entire contents of which are incorporated herein by reference). Thus, immunization with heteroclitic epitopes offers the capacity to modulate cytokine production by induction of specific subsets of effector T cells, thereby altering the course of disease.

[0203] In some embodiments, heteroclitic epitopes offer an advantage in drug development since significantly smaller amounts of peptide are needed for treatment doses, due to their strong biological potency. This feature overcomes certain manufacturing and toxicity concerns. In this regard, it has been shown that a heteroclitic analog of a MART-1 peptide (Rivoltini, et ah, Cancer Research 59:301 (1999), the entire contents of which are incorporated herein by reference), which generated antigen specific T cells in melanoma patients, was active at much lower concentrations than the native epitope. Similar results were reported by Schlom and colleagues (Zaremba, et ah, Cancer Research 57:4570 (1997), the entire contents of which are incorporated herein by reference) regarding heteroclitic analog of the CEA derived CAP1 epitope.

Nucleobase editors

[0204] The methods of generating immunogenic peptides or epitopes from tumor associated antigens as cancer vaccines described herein, are enabled by the use of the nucleobase editors. As described herein, a nucleobase editor is a fusion protein comprising: (i) a programmable DNA binding protein domain; and (ii) a deaminase domain. It is to be understood that any programmable DNA binding domain may be used in the based editors.

[0205] In some embodiments, the programmable DNA binding protein domain comprises the DNA binding domain of a zinc finger nuclease (ZFN) or a transcription activator-like effector domain (TALE). In some embodiments, the programmable DNA binding protein domain may be programmed by a guide nucleotide sequence, and is thus referred as a "guide nucleotide sequence-programmable DNA binding-protein domain." In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Cas9, or dCas9. A dCas9, as used herein, encompasses a Cas9 that is completely inactive in its nuclease activity, or partially inactive in its nuclease activity {e.g., a Cas9 nickase). Thus, in some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a Cas9 nickase. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Cpfl. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Argonaute. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive CasX or CasY, e.g., as described in Burstein et al., New CRISPR-Cas systems from uncultivated microbes, Nature 542, 237-241, 2017, incorporated herein by reference.

[0206] In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a dCas9 domain. In some embodiments, the guide nucleotide sequence- programmable DNA binding protein is a Cas9 nickase. In some embodiments, the dCas9 domain comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein (e.g., SEQ ID NOs: 11-260), and comprises mutations corresponding to D10X (X is any amino acid except for D) and/or H840X (X is any amino acid except for H) in SEQ ID NO: 1. In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein (e.g. , SEQ ID NOs: 11-260), and comprises mutations corresponding to D10A and/or H840A in SEQ ID NO: 1. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein (e.g. , SEQ ID NOs: 11-260), and comprises mutations

corresponding to D10X (X is any amino acid except for D) in SEQ ID NO: 1 and a histidine at a position correspond to position 840 in SEQ ID NO: 1. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein (e.g., SEQ ID NOs: 11-260), and comprises mutations corresponding to D10A in SEQ ID NO: 1 and a histidine at a position correspond to position 840 in SEQ ID NO: 1. In some embodiments, variants or homologues of dCas9 or Cas9 nickase (e.g., variants of SEQ ID NO: 2 or SEQ ID NO: 3, respectively) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to SEQ ID NO: 2 or SEQ ID NO: 3, respectively, and comprises mutations corresponding to D10A and/or H840A in SEQ ID NO: 1. In some embodiments, variants of Cas9 (e.g., variants of SEQ ID NO: 2) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 2, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more, provided that the dCas9 variants comprise mutations corresponding to DIOA and/or H840A in SEQ ID NO: 1. In some embodiments, variants of Cas9 nickase (e.g., variants of SEQ ID NO: 3) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 3, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more, provided that the dCas9 variants comprise mutations corresponding to DIOA and comprises a histidine at a position corresponding to position 840 in SEQ ID NO: 1.

[0207] Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et ah , Nature Biotechnology. 2013; 31(9): 833-838, which are incorporated herein by reference), or K603R (See, e.g. , Chavez et ah , Nature Methods 12, 326-328, 2015, which is incorporated herein by reference.

[0208] In some embodiments, the nucleobase editors utilized in the present invention comprise a Cas9 domain with decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, the nucleobase editors described herein comprises a dCas9 (e.g., with DIOA and H840A mutations) or a Cas9 nickase (e.g. , with DIOA mutation), wherein the dCas9 or the Cas9 nickase further comprises one or more of a N497X, R661X, Q695X, and/or Q926X mutation of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the nucleobase editors described herein comprises a dCas9 (e.g. , with DIOA and H840A mutations) or a Cas9 nickase (e.g., with DIOA mutation), wherein the dCas9 or the Cas9 nickase further comprises one or more of a N497A, R661A, Q695A, and/or Q926A mutation of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11- 260. In some embodiments, the dCas9 domain (e.g. , of any of the nucleobase editors provided herein) comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9. In some embodiments, the nucleobase editor comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 293-302 and 321.

Cas9 variant with decreased electrostatic interactions between the Cas9 and DNA backbone

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARR

RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA YHEKYPTIYHLRK

KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAK

AILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL SKDTYDDDLDNLL

AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK ALVRQQLPEKYK

EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGE

LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASA

QSFIERMTAFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQK KAIVDLLFKTN

RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE DILEDIVLTLTLFE

DREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLK SDGFANRNFM

ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM GRHKPENIVIEM

ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGR DMYVDQELDIN

RLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLN AKLITQRKFD

NLTKAERGGLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKV ITLKSKLVSDFR

KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATA

KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ VNIVKKTEVQTG

GFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERS

SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASH

YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIH

LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 9, mutations relative to SEQ ID NO: 1 are bolded and underlined)

High fidelity nucleobase editor

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK HVEVNFIEKF

TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRN RQGLRDLISSGVTI

QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRR KQPQLTFFTIALQ

SCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDE YKVPSKKFKVLG

NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFL

VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLN

PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALS

LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITK

APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEE FYKFIKPILEKM

DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGP

LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKH SLLYEYFTVYN

ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE ISGVEDRFNASL GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWG

ALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGS

PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG IKELGSQILKEH

PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKS

DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RAITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV VGTALIKKYPK

LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR PLIETNGETGEIV

WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVA

YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGR

KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKR

VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRY TSTKEVLDATLIH

QSITGLYETRIDLSQLGGD (SEQ ID NO: 321)

[0209] In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpfl, C2cl, C2c2, and C2c3.

Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. Cas9 and Cpfl are Class 2 effectors. In addition to Cas9 and Cpfl, three distinct Class 2 CRISPR-Cas systems (C2cl, C2c2, and C2c3) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems", Mol. Cell, 2015 Nov 5; 60(3): 385-397, the entire contents of which are herein incorporated by reference. Effectors of two of the systems, C2cl and C2c3, contain RuvC- like endonuclease domains related to Cpfl . A third system, C2c2 contains an effector with two predicted HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA- independent, unlike production of CRISPR RNA by C2cl. C2cl depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single- stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpfl. See, e.g., East-Seletsky, et al., "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection", Nature, 2016 Oct 13 ;538(7624) :270-273, the entire contents of which are hereby

incorporated by reference. In vitro biochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector," Science, 2016 Aug 5;

353(6299), the entire contents of which are hereby incorporated by reference.

[0210] The crystal structure of Alicyclobaccillus acidoterrastris C2cl (AacC2cl) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., "C2cl-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism", Mol. Cell, 2017 Jan 19;65(2):310-322, incorporated herein by reference. The crystal structure has also been reported for Alicyclobacillus acidoterrestris C2cl bound to target DNAs as ternary complexes. See, e.g., Yang et al., "PAM-dependent Target DNA

Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease", Cell, 2016 Dec

15;167(7): 1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2cl, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2cl -mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2cl ternary complexes and previously identified Cas9 and Cpf 1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.

[0211] In some embodiments, the nucleobase editors described herein comprise a C2cl, a C2c2, or a C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a C2cl protein. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a C2c2 protein. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2cl, C2c2, or C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a naturally-occurring C2cl, C2c2, or C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 1057-1059. In some embodiments, the guide nucleotide sequence- programmable DNA binding protein comprises an amino acid sequence of any one SEQ ID NOs: 1057-1059. It should be appreciated that C2cl, C2c2, or C2c3 from other bacterial species may also be used in accordance with the present disclosure. [0212] C2c 1 (uniprot.org/uniprot/T0D7 A2#) splT0D7 A2IC2C 1_ALIAG CRISPR- associated endonuclease C2cl OS=Alicyclobacillus acidoterrestris (strain ATCC 49025 /

DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2cl PE=1 SV=1

MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQE CDKTAE

ECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARK FLSPLADKDAV

GGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLM RVYTDSEMS

SVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKNF VGQEHLV

HLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAE IKNVQRRNT

RRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATA HPIWTRFDKLG

GNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDP NEPIALYFRDYG

AEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAA VFRLVGDNH

RAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDEL KPNSKGRVPFFF

PIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCG SEDVGRRERSW

AKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMG KQVRDWRK

DVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSR FAITLREHIDHAK

EDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPS ENNQLMQWSH

RGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFP WWLNKFVVEHT

LDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQI RLRCDWGEVDGE

LVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVE ADEAREKSVVL

MRDPSGIINRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQDSACENTGDI (SEQ ID NO: 1057)

[0213] C2c2 (uniprot.org/uniprot/P0DOC6) >splP0DOC6IC2C2_LEPSD CRISPR- associated endoribonuclease C2c2 OS=Leptotrichia shahii (strain DSM 19757 / CCUG

47503 / CIP 107916 / JCM 16776 / LB37) GN=c2c2 PE=1 SV=1

MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYIN YKKNDNI

LKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGIT KKKIIDEAIRQGITK

DDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNI NMSLYKIIEKIIENETEK

VFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKK SKNKKMLVEKIL

NINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKH EKFKIERENKKDK

IVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKV NFDSKKFSKKSDEEK

ELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLE HIMYLGKLRHNDID

MTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREK NYVLDKKILNSKI

KIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINI IQNLKISDEEVSKAL

NLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDT IETEKIVLNALIYVN

KELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNN KAIKKYQKKVIECY

IGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEY IISIFALLNSNAVIN

KIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEI EKDFDDFKIQTKKE

IFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQR KLSNINKKDLKKKV

DQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKER KNELYIYKKNLFLNI

GNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKE YKEKYIKKLKEND DFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFER DMHYIVNGL

RELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDL SENSEINKPENESIR

NYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDE LKKKFKLIGNNDI

LERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL (SEQ ID NO: 1058)

[0214] C2c3, translated from >CEPX01008730.1 marine metagenome genome assembly

TARA_037_MES_0.1-0.22, contig TARA_037_MES_0.1-0.22_scaffold22115_l, whole genome shotgun sequence.

MRSNYHGGRNARQWRKQISGLARRTKETVFTYKFPLETDAAEIDFDKAVQTYGIAEGVGH GSLIGLVC

AFHLSGFRLFSKAGEAMAFRNRSRYPTDAFAEKLSAIMGIQLPTLSPEGLDLIFQSP PRSRDGIAPVWSE

NEVRNRLYTNWTGRGPANKPDEHLLEIAGEIAKQVFPKFGGWDDLASDPDKALAAAD KYFQSQGDFP

SIASLPAAIMLSPANSTVDFEGDYIAIDPAAETLLHQAVSRCAARLGRERPDLDQNK GPFVSSLQDALVS

SQNNGLSWLFGVGFQHWKEKSPKELIDEYKVPADQHGAVTQVKSFVDAIPLNPLFDT THYGEFRASVA

GKVRSWVANYWKRLLDLKSLLATTEFTLPESISDPKAVSLFSGLLVDPQGLKKVADS LPARLVSAEEAI

DRLMGVGIPTAADIAQVERVADEIGAFIGQVQQFNNQVKQKLENLQDADDEEFLKGL KIELPSGDKEPP

AINRISGGAPDAAAEISELEEKLQRLLDARSEHFQTISEWAEENAVTLDPIAAMVEL ERLRLAERGATGD

PEEYALRLLLQRIGRLANRVSPVSAGSIRELLKPVFMEEREFNLFFHNRLGSLYRSP YSTSRHQPFSIDVG

KAKAIDWIAGLDQISSDIEKALSGAGEALGDQLRDWINLAGFAISQRLRGLPDTVPN ALAQVRCPDDVR

IPPLLAMLLEEDDIARDVCLKAFNLYVSAINGCLFGALREGFIVRTRFQRIGTDQIH YVPKDKAWEYPDR

LNTAKGPINAAVSSDWIEKDGAVIKPVETVRNLSSTGFAGAGVSEYLVQAPHDWYTP LDLRDVAHLVT

GLPVEKNITKLKRLTNRTAFRMVGASSFKTHLDSVLLSDKIKLGDFTIIIDQHYRQS VTYGGKVKISYEP

ERLQVEAAVPVVDTRDRTVPEPDTLFDHIVAIDLGERSVGFAVFDIKSCLRTGEVKP IHDNNGNPVVGT

VAVPSIRRLMKAVRSHRRRRQPNQKVNQTYSTALQNYRENVIGDVCNRIDTLMERYN AFPVLEFQIKN

FQAGAKQLEIVYGS (SEQ ID NO: 1059)

[0215] Cas9 recognizes a short motif (PAM motif) in the CRISPR repeat sequences in the target DNA sequence. A "PAM motif," or "protospacer adjacent motif," as used herein, refers a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Naturally, Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component (not found in the bacterial genome) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.

[0216] Wild-type Streptococcus pyogenes Cas9 recognizes a canonical PAM sequence (5'- NGG-3')- Other Cas9 nucleases (e.g., Cas9 from Streptococcus thermophiles, Staphylococcus aureus, Neisseria meningitidis, or Treponema denticolaor) and Cas9 variants thereof have been described in the art to have different, or more relaxed PAM requirements. For example, in Kleinstiver et al, Nature 523, 481-485, 2015; Klenstiver et al, Nature 529, 490-495, 2016; Ran et al, Nature, Apr 9; 520(7546): 186-191, 2015; Kleinstiver et al, Nat

Biotechnol, 33(12): 1293-1298, 2015; Hou et al, Proc Natl Acad Sci U S A, 110(39): 15644-9, 2014; Prykhozhij et al, PLoS One, 10(3): eOl 19372, 2015; Zetsche et al, Cell 163, 759-771, 2015; Gao et al, Nature Biotechnology, doi: 10.1038/nbt.3547, 2016; Want et al, Nature 461, 754-761, 2009; Chavez et al, doi: dx.doi.org/10.1101/058974; Fagerlund et al, Genome Biol. 2015; 16: 25, 2015; Zetsche et al, Cell, 163, 759-771, 2015; and Swarts et al, Nat Struct Mol Biol, 21(9):743-53, 2014, each of which is incorporated herein by reference.

[0217] Thus, the guide nucleotide sequence-programmable DNA-binding protein of the present disclosure may recognize a variety of PAM sequences including, without limitation: NGG, NGAN, NGNG, NGAG, NGCG, NNGRRT, NGRRN, NNNRRT, NNNGATT, NNAGAAW, NAAAC, TTN, TTTN, and YTN, wherein Y is a pyrimidine, and N is any nucleobase. In some embodiments, the PAM is located 5' of the target base. In some embodiments, the PAM is located 3 ' of the target base.

[0218] One example of an RNA-programmable DNA-binding protein that has different PAM specificity is Clustered Regularly Interspaced Short Palindromic Repeats from

Prevotella and Francisella 1 (Cpfl). Similar to Cas9, Cpfl is also a class 2 CRISPR effector. It has been shown that Cpflmediates robust DNA interference with features distinct from Cas9. Cpfl is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double- stranded break. Out of 16 Cpfl-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells.

[0219] Also useful in the present disclosure are nuclease-inactive Cpfl (dCpfl) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al, Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpfl is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpfl nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpfl (SEQ ID NO: 10) inactivates Cpfl nuclease activity. In some embodiments, the dCpfl of the present disclosure comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 10. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions that inactivates the RuvC domain of Cpfl may be used in accordance with the present disclosure.

[0220] Thus, in some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Cpfl (dCpfl). In some embodiments, the dCpfl comprises the amino acid sequence of any one SEQ ID NOs: 261-267. In some embodiments, the dCpfl comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to SEQ ID NO: 10, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A,

E1006A/D1255A, or D917A/ E1006A/D1255A in SEQ ID NO: 10. Cpfl from other bacterial species may also be used in accordance with the present disclosure.

Wild type Francisella novicida Cpfl (SEQ ID NO: 10) (D917, E1006, and D1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

DANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN Francisella novicida Cpfl D917A (SEQ ID NO: 261) (A917, E1006, and D1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

DANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Francisella novicida Cpfl E1006A (SEQ ID NO: 262) (D917, A1006, and D1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQE FFSKFDKICY NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKL LKDYSIEY GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQ APKNMPQDA DANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Francisella novicida Cpfl D1255A (SEQ ID NO: 263) (D917, E1006, and A1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

AANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Francisella novicida Cpfl D917A/E1006A (SEQ ID NO: 264) (A917, A1006, and D1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQG YKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

DANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Francisella novicida Cpfl D917A/D1255A (SEQ ID NO: 265) (A917, E1006, and A1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

AANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Francisella novicida Cpfl E1006A/D1255A (SEQ ID NO: 266) (D917, A1006, and A1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKK GQESDLILW LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIY RIVDDNLPKFLE NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYL NQSGITKFNT IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLE DDSDVVTTMQ SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVI GTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

AANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Francisella novicida Cpfl D917A/E1006A/D1255A (SEQ ID NO: 267) (A917, A1006, and A 1255 are bolded and underlined)

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCIS

EDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID AKKGQESDLILW

LKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTS IIYRIVDDNLPKFLE

NKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFN NYLNQSGITKFNT

IIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVID KLEDDSDVVTTMQ

SFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY SVIGTAVLEYITQ

QIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQ

NKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDK ANILDKDEHFYL

VFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGV

MNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDIL RIRNHSTHTKNG

SPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE NQGYKLTFENISES

YIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKK

ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFN DEINLLLKEKAND

VHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSA RKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDK

TGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSK SQEFFSKFDKICY

NLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKEL EKLLKDYSIEY

GHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFD SRQAPKNMPQDA

AANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

[0221] In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a Cpfl protein from a Acidaminococcus species (AsCpfl). Cpfl proteins form Acidaminococcus species have been described previously and would be apparent to the skilled artisan. Exemplary Acidaminococcus Cpfl proteins (AsCpfl) include, without limitation, any of the AsCpfl proteins provided herein.

[0222] Wild-type AsCpfl- Residue R912 is indicated in bold underlining and residues 661-667 are indicated in italics and underlining.

TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQCLQLVQL

DWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEI YKGLFKAELFNG

KVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRL

ITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISRE AGTEKIKGLNEVLNL

AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNEL

NSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSL KHEDINLQEIISAAG

KELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEF

SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNG AILFVKNGLY

YLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNF

IEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTK TTSIDLSSLRPSSQ

YKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNL HTLYWTGLFSPE

NLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDY VNHRLSHDLS

DEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYL KEHPETPIIGIDRGE

RNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLK QGYLSQVIHEIVD

LMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGG VLNPYQLTDQ

FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLH YDVKTGDFILHFK

MNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRD LYPANELIALLE

EKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDL NGVCFDSRFQNP

EWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 1060)

[0223] AsCpfl (R912A)- Residue A912 is indicated in bold underlining and residues 661-

667 are indicated in italics and underlining.

TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQCLQLVQL

DWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEI YKGLFKAELFNG

KVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRL

ITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISRE AGTEKIKGLNEVLNL

AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNEL

NSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSL KHEDINLQEIISAAG

KELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEF

SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNG AILFVKNGLY

YLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNF

IEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTK TTSIDLSSLRPSSQ

YKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNL HTLYWTGLFSPE

NLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDY VNHRLSHDLS

DEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYL KEHPETPIIGIDRGE ANLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGY LSQVIHEIVD

LMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGG VLNPYQLTDQ

FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLH YDVKTGDFILHFK

MNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRD LYPANELIALLE

EKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDL NGVCFDSRFQNP

EWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 1061)

[0224] In some embodiments, the nucleic acid programmable DNA binding protein is a Cpfl protein from a Lachnospiraceae species (LbCpfl). Cpfl proteins form Lachnospiraceae species have been described previously have been described previously and would be apparent to the skilled artisan. Exemplary Lachnospiraceae Cpfl proteins (LbCpfl) include, without limitation, any of the LbCpfl proteins provided herein.

[0225] Wild-type LbCpfl

MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLS FINDVLHSI

KLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIET ILPEFLDDKDEIAL

VNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIF DKHEVQEIKEKILN

SDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQ KLPKFKPLYKQVL

SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFV KNGPAISTISKDIFG

EWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSV VEKLKEIIIQK

VDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGD

FVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATI LRYGSKYYLAI

MDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQK IYKNGTFKK

GDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKV SFESASKKEVD

KLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRR ASLKKEELVVH

PANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEV RVLLKHDDNPYVI

GIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEA RQNWTSIENIKELK

AGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYM VDKKSNPCAT

GGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADS KKFISSFDRIMYVP

EEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAY KELFNKYGINY

QQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYD SRNYEAQENAIL

PKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH (SEQ ID NO:

1062)

[0226] LbCpfl (R836A)

MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLS FINDVLHSI KLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILP EFLDDKDEIAL VNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKH EVQEIKEKILN SDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLP KFKPLYKQVL

SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFV KNGPAISTISKDIFG

EWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSV VEKLKEIIIQK

VDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGD

FVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATI LRYGSKYYLAI

MDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQK IYKNGTFKK

GDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKV SFESASKKEVD

KLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRR ASLKKEELVVH

PANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEV RVLLKHDDNPYVI

GIDRGEANLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEA RQNWTSIENIKEL

KAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNY MVDKKSNPCA

TGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIAD SKKFISSFDRIMYV

PEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSA YKELFNKYGIN

YQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFY DSRNYEAQENAI

LPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH (SEQ ID NO:

1063)

[0227] LbCpfl (R1138A)

MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLS FINDVLHSI

KLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIET ILPEFLDDKDEIAL

VNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIF DKHEVQEIKEKILN

SDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQ KLPKFKPLYKQVL

SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFV KNGPAISTISKDIFG

EWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSV VEKLKEIIIQK

VDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGD

FVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATI LRYGSKYYLAI

MDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQK IYKNGTFKK

GDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKV SFESASKKEVD

KLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRR ASLKKEELVVH

PANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEV RVLLKHDDNPYVI

GIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEA RQNWTSIENIKELK

AGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYM VDKKSNPCAT

GGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADS KKFISSFDRIMYVP

EEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAY KELFNKYGINY

QQGDIRALLCEQSDKAFYSSFMALMSLMLQMANSITGRTDVDFLISPVKNSDGIFYD SRNYEAQENAIL

PKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH (SEQ ID NO:

1064) [0228] In some embodiments, the Cpfl protein is a crippled Cpfl protein. As used herein a "crippled Cpfl" protein is a Cpfl protein having diminished nuclease activity as compared to a wild-type Cpfl protein. In some embodiments, the crippled Cpfl protein preferentially cuts the target strand more efficiently than the non-target strand. For example, the Cpfl protein preferentially cuts the strand of a duplexed nucleic acid molecule in which a nucleotide to be edited resides. In some embodiments, the crippled Cpfl protein

preferentially cuts the non-target strand more efficiently than the target strand. For example, the Cpfl protein preferentially cuts the strand of a duplexed nucleic acid molecule in which a nucleotide to be edited does not reside. In some embodiments, the crippled Cpfl protein preferentially cuts the target strand at least 5% more efficiently than it cuts the non-target strand. In some embodiments, the crippled Cpfl protein preferentially cuts the target strand at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 100% more efficiently than it cuts the non-target strand.

[0229] In some embodiments, a crippled Cpfl protein is a non-naturally occurring Cpfl protein. In some embodiments, the crippled Cpfl protein comprises one or more mutations relative to a wild-type Cpfl protein. In some embodiments, the crippled Cpfl protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type Cpfl protein. In some embodiments, the crippled Cpfl protein comprises an R836A mutation mutation as set forth in SEQ ID NO: 763, or in a

corresponding amino acid in another Cpfl protein. It should be appreciated that a Cpfl comprising a homologous residue (e.g., a corresponding amino acid) to R836A of SEQ ID NO: 763 could also be mutated to achieve similar results. In some embodiments, the crippled Cpfl protein comprises a Rl 138A mutation as set forth in SEQ ID NO: 763, or in a corresponding amino acid in another Cpfl protein. In some embodiments, the crippled Cpfl protein comprises an R912A mutation mutation as set forth in SEQ ID NO: 762, or in a corresponding amino acid in another Cpfl protein. Without wishing to be bound by any particular theory, residue R838 of SEQ ID NO: 763 (LbCpfl) and residue R912 of SEQ ID NO: 762 (AsCpfl) are examples of corresponding (e.g., homologous) residues. For example, a portion of the alignment between SEQ ID NO: 762 and 763 shows that R912 and R838 are corresponding residues.

[0230] In some embodiments, any of the Cpfl proteins provided herein comprises one or more amino acid deletions. In some embodiments, any of the Cpfl proteins provided herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions. Without wishing to be bound by any particular theory, there is a helical region in Cpfl, which includes residues 661-667 of AsCpfl (SEQ ID NO: 762), that may obstruct the function of a deaminase (e.g., APOBEC) that is fused to the Cpfl. This region comprises the amino acid sequence KKTGDQK. Accordingly, aspects of the disclosure provide Cpfl proteins comprising mutations (e.g., deletions) that disrupt this helical region in Cpfl. In some embodiments, the Cpfl protein comprises one or more deletions of the following residues in SEQ ID NO: 762, or one or more corresponding deletions in another Cpfl protein: K661, K662, T663, G664, D665, Q666, and K667. In some embodiments, the Cpfl protein comprises a T663 and a D665 deletion in SEQ ID NO: 762, or corresponding deletions in another Cpfl protein. In some embodiments, the Cpfl protein comprises a K662,T663, D665, and Q666 deletion in SEQ ID NO: 762, or corresponding deletions in another Cpfl protein. In some embodiments, the Cpfl protein comprises a K661, K662, T663, D665, Q666 and K667 deletion in SEQ ID NO: 762, or corresponding deletions in another Cpfl protein.

[0231] AsCpfl (deleted T663 and D665)

TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQCLQLVQL

DWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEI YKGLFKAELFNG

KVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRL

ITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISRE AGTEKIKGLNEVLNL

AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNEL

NSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSL KHEDINLQEIISAAG

KELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEF

SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNG AILFVKNGLY

YLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNF

IEPLEITKEIYDLNNPEKEPKKFQTAYAKKGQKGYREALCKWIDFTRDFLSKYTKTT SIDLSSLRPSSQY

KDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLH TLYWTGLFSPEN

LAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYV NHRLSHDLSD

EARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLK EHPETPIIGIDRGER

NLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQ GYLSQVIHEIVDL

MIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGV LNPYQLTDQF

TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHY DVKTGDFILHFK

MNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRD LYPANELIALLE

EKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDL NGVCFDSRFQNP

EWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 1065)

[0232] AsCpfl (deleted K662, T663, D665, and Q666) TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQCLQLVQL

DWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEI YKGLFKAELFNG

KVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRL

ITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISRE AGTEKIKGLNEVLNL

AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNEL

NSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSL KHEDINLQEIISAAG

KELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEF

SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNG AILFVKNGLY

YLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNF

IEPLEITKEIYDLNNPEKEPKKFQTAYAKGKGYREALCKWIDFTRDFLSKYTKTTSI DLSSLRPSSQYKDL

GEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLY WTGLFSPENLAK

TSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHR LSHDLSDEAR

ALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP ETPIIGIDRGERNLI

YITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYL SQVIHEIVDLMI

HYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLN PYQLTDQFTS

FAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV KTGDFILHFKMN

RNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLY PANELIALLEEKG

IVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGV CFDSRFQNPEWP

MDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 1066)

[0233] AsCpfl (deleted K661, K662, T663,D665, Q666, and K667)

TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQCLQLVQL

DWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEI YKGLFKAELFNG

KVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDN FPKFKENCHIFTRL

ITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISRE AGTEKIKGLNEVLNL

AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNEL

NSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSL KHEDINLQEIISAAG

KELSEAFKQKTSEILSHAHAALDQPLPTTMLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEF

SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNG AILFVKNGLY

YLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNF

IEPLEITKEIYDLNNPEKEPKKFQTAYAGGYREALCKWIDFTRDFLSKYTKTTSIDL SSLRPSSQYKDLGE

YYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWT GLFSPENLAKTS

IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS HDLSDEARAL

LPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPET PIIGIDRGERNLIYIT

VIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQV IHEIVDLMIHYQ

AVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQ LTDQFTSFAK

MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTG DFILHFKMNRNL

SFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPAN ELIALLEEKGIVF

RDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFD SRFQNPEWPMD

ADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 1067) [0234] In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain of the present disclosure has no requirements for a PAM sequence. One example of such guide nucleotide sequence-programmable DNA-binding protein may be an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5' phosphorylated ssDNA of -24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the codons that may be targeted. The characterization and use of NgAgo have been described in Gao et ah, Nat Biotechnol. Epub 2016 May 2. PubMed PMID: 27136078; Swarts et al, Nature. 507(7491) (2014):258-61; and Swarts et ah, Nucleic Acids Res. 43(10) (2015):5120-9, each of which are incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided in SEQ ID NO: 270.

Wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 270)

MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFEQDNGERRYITL

WKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTTVENATAQEVGTTD

EDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLHTYTLT

ATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDLDCG

VRVE ADETRTLD YTT AKDRLLARELVEEGLKRS LWDD YLVRGIDE VLS KEPVLTCD

EFDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPR

RGHIVWGLRDECATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRR

QGHGDD A VS FPQELL A VEPNTHQIKQFAS D GFHQQ ARS KTRLS AS RC S EKAQ AF AER

LDPVRLNGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPDETFSKGIV N

PPES FE V A V VLPEQQ ADTC KAQWDTM ADLLNQ AG APPTRS ET VQ YD AFS S PES IS LN

VAGAIDPSEVDAAFVVLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRF

RDAKIFYTRNVALGLLAAAGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINI

AATATAVYKDGTILGHSSTRPQLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIH R

DGFMNEDLDPATEFLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNE P

RATVATFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNST A

RLPITTAYADQASTHATKGYLVQTGAFESNVGFL [0235] Also provided herein are Cas9 variants that have relaxed PAM requirements (PAMless Cas9). PAMless Cas9 exhibits an increased activity on a target sequence that does not include a canonical PAM (e.g. , NGG) at its 3'-end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 1, e.g., increased activity by at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000- fold, or at least 1,000,000-fold. Such Cas9 variants that have relaxed PAM requirements are described in US Provisional Applications 62/245828, 62/279346, 62/311763, 62/322178, and 62/357,332, each of which is incorporated herein by reference. In some embodiments, the dCas9 or Cas9 nickase useful in the present disclosure may further comprise mutations that relax the PAM requirements, e.g. , mutations that correspond to A262T, K294R, S409I, E480K, E543D, M694I, or E1219V in SEQ ID NO: 1.

[0236] Other on-limiting, exemplary Cas9 variants (including dCas9, Cas9 nickase, and Cas9 variants with alternative PAM requirements) suitable for use in the nucleobase editors described herein and their respective sequence are provided below.

VREPv-nCas9 ( D 1 OA/D 1 135 V/Ci 12 1 8R/R 13351-71 ^" 1337R ) .V. pyogenes Cas9 Nickase

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEV AYHEKYPTIYHLR

KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDA

KAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDN

LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEK

YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHL

GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGA

SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFK

TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLT

LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRN

FMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVK VMGRHKPENIVI

EMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLON GRDMYVDQEL

DINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRO LLNAKLITQRK

FDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSD

FRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEOEIGKA

TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM POVNIVKKTEVO

1GGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIME

RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELA LPSKYVNFLYLA

SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO:

821) (single underline: HNH domain; double underline: RuvC domain)

VQR-nCas9 (D10A/D1135V/R1335Q/T1337R) S. pyogenes Cas9 Nickase

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEV AYHEKYPTIYHLR

KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDA

KAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDN

LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEK

YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHL

GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGA

SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFK

TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLT

LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRN

FMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVK VMGRHKPENIVI

EMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLON GRDMYVDQEL

DINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRO LLNAKLITQRK

FDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSD

FRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEOEIGKA

TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM POVNIVKKTEVO

1GGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIME

RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLA

SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENI

IHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO:

822) (single underline: HNH domain; double underline: RuvC domain)

EQR-nCas9 (D10A/D1135E/R1335Q/T1337R) S. pyogenes Cas9 Nickase

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEV AYHEKYPTIYHLR

KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF EENPINASGVDA

KAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDN

LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEK

YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHL

GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGA

SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFK

TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLT

LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRN

FMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVK VMGRHKPENIVI

EMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLON GRDMYVDQEL DINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWROLLN AKLITQRK

FDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSD

FRKDFOFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEOEIGKA

TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM POVNIVKKTEVO

1GGFSKESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVK ELLGITIME

RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLA

SHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENI

IHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLG GD (SEQ ID NO:

823) (single underline: HNH domain; double underline: RuvC domain)

KKH-nCas9 (D10A/E782K/N968K/R1015H) S. aureus Cas9 Nickase

MKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRR RHRIQRVKK

LLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQI

SRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQ SFIDTYIDLLE

TRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLN NLVITRDENE

KLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYH DIKDITARKEIIEN

AELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIA

IFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPND IIIELAREKNSKDAQK

MINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDL LNNPFNYEVDHIIP

RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGR ISKTKKEYLLEER

DINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRK WKFKKERNKGY

KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFI TPHQIKHIKDFK

DYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDP

QTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHL DITDDYPNSR

NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISN QAEFIASFYKN

DLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQS IKKYSTDILGNLYE

VKSKKHPQIIKKG (SEQ ID NO:268)

Streptococcus thermophilus CRISPR1 Cas9 (StlCas9) Nickase (D9A)

MSDLVLGLAIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLTRRKK HRRVRLNRL

FEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASD DGNSSIGDYAQIVK

ENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRIL QTQQEFNPQITDE

FINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR AAKASYTAQEFNL

LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGY RIDKSGKAEIHT

FEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQV DELVQFRKANS

SIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLT EEIYNPVVAKSVR

QAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQ YNGKAELPHSV

FHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLA NKVLVYATANQE

KGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIE RNLVDTRYASR VVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLN LWKKQKN

TLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDS KFNRKISDATIYAT

RQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIE PILENYPNKQI

NEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNN KVVLQSVSPWR

ADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTL YKNDLLLVKDT

ETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGK SNISIYKVRTD

VLGNQHIIKNEGDKPKLDF (SEQ ID NO: 269)

Streptococcus thermophilus CRISPR3Cas9 (St3Cas9) Nickase (D10A)

MTKPYSIGLAIGTNSVGWAVITDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAE GRRLKRTA

RRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEE KVYHDEFPTIYHL

RKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAI FESDLSLENSKQ

LEEIVKDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCFNLDEKASL HFSKESYDEDLETL

LGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYNEHKEDLALL KEYIRNISLKTYN

EVFKDDTKNGYAGYIDGKTNQEDFYVYLKNLLAEFEGADYFLEKIDREDFLRKQRTF DNGSIPYQIHLQ

EMRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKIT PWNFEDVIDKESS

AEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSKQ KKDIVRLYFKDK

RKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEA IIEEIIHTLTIFEDRE

MIKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDG ISNRNFMQLIHDD

ALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRK PESIVVEMARENQ

YTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNG KDMYTGDDLDI

DRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDFPSLEVVKKRKTFWYQLL KSKLISQRKFDNL

TKAERGGLLPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRAVRTVKIIT LKSTLVSQFRKD

FELYKVREINDFHHAHDAYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERKSAT EKVYFYSNIMNI

FKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQN HGLDRGKPKGL

FNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKKKITN VLEFQGISILDRI

NYRKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGN QIFLSQKFVKLLYH

AKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQSWQNH SIDELCSSFIGPT

GSERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRID LAKLGEG (SEQ ID

NO: 824)

[0237] In some embodiments, the nucleobase editors useful in the present disclosure comprises: (i) a guide nucleotide sequence-programmable DNA-binding protein domain; and (ii) a deaminase domain. In some embodiments, the deaminase domain of the fusion protein is a cytosine deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is a rat APOBEC1. In some embodiments, the deaminase is a human APOBEC1. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an

APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some

embodiments, the deaminase is a Lamprey CDA1 (pmCDAl). In some embodiments, the deaminase is a human APOBEC3G or a functional fragment thereof. In some embodiments, the deaminase is an APOBEC3G variant comprising mutations correspond to the

D316R/D317R mutations in the human APOBEC3G. Exemplary, non-limiting cytosine deaminase sequences that may be used in accordance with the methods of the present disclosure are provided in Example 1 below.

[0238] In some embodiments, the cytosine deaminase is a wild type deaminase or a deaminase as set forth in SEQ ID NOs: 271-292, 303, and 1072-1083. In some

embodiments, the cytosine deaminase domains of the fusion proteins provided herein include fragments of deaminases and proteins homologous to a deaminase. For example, in some embodiments, a deaminase domain may comprise a fragment of the amino acid sequence set forth in any of SEQ ID NOs: 271-292, 303, and 1072-1083. In some embodiments, a deaminase domain comprises an amino acid sequence homologous to the amino acid sequence set forth in any of SEQ ID NOs: 271-292, 303, and 1072-1083, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in any of SEQ ID NOs: 271-292, 303, and 1072-1083. In some embodiments, proteins comprising a deaminase, a fragments of a deaminase, or homologs of a deaminase or a deaminase are referred to as "deaminase variants." A deaminase variant shares homology to a deaminase, or a fragment thereof. For example a deaminase variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to a wild type deaminase or a deaminase as set forth in any of SEQ ID NOs: 271-292, 303, and 1072-1083. In some embodiments, the deaminase variant comprises a fragment of the deaminase, such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to the corresponding fragment of wild type deaminase or a deaminase as set forth in any of SEQ ID NOs: 271-292, 303, and 1072-1083. In some embodiments, the cytosine deaminase is at least at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to an APOBEC3G variant as set forth in SEQ ID NO: 291 or SEQ ID NO: 292, and comprises mutations corresponding to the D316E/D317R mutations in SEQ ID NO: 290.

[0239] In some embodiments, the cytosine deaminase domain is fused to the N-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain. For example, the fusion protein may have an architecture of NH ₂ -[cytosine deaminase]-[ guide nucleotide sequence-programmable DNA-binding protein domain] -COOH. The "]-[" used in the general architecture above indicates the presence of an optional linker sequence. The term "linker," as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g. , two domains of a fusion protein, such as, for example, a dCas9 domain and a cytosine deaminase domain. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g. , a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5- 100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100- 150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0240] In some embodiments, the cytosine deaminase domain and the Cas9 domain are fused to each other via a linker. Various linker lengths and flexibilities between the deaminase domain (e.g., APOBEC 1) and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGS) _n (SEQ ID NO: 337), (GGGGS) _n (SEQ ID NO: 308), (GGS)n (SEQ ID NO: 784), and (G) _n (SEQ ID NO: 783)to more rigid linkers of the form (EAAAK) _n (SEQ ID NO: 309), SGSETPGTSESATPES (SEQ ID NO: 310) (see, e.g. , Guilinger et ah , Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference), (SGGS)„SGSETPGTSESATPES(SGGS)„ (SEQ ID NO: 1068), (XP)„ (SEQ ID NO: 785), or a combination of any of these, wherein X is any amino acid and n is independently an integer between 1 and 30, in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. In some embodiments, the linker comprises a (GGS) _n (SEQ ID NO: 784) motif, wherein n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the linker comprises a (GGS) _n (SEQ ID NO: 784) motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 310), also referred to as the XTEN linker. In some embodiments, the linker comprises an amino acid sequence chosen from the group including, but not limited to, AGVF (SEQ ID NO: 825), GFLG (SEQ ID NO: 826), FK, AL, ALAL (SEQ ID NO: 827), or ALALA (SEQ ID NO: 828). In some embodiments, suitable linker motifs and configurations include those described in Chen et ah , Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69, which is incorporated herein by reference. In some embodiments, the linker may comprise any of the following amino acid sequences:

VPFLLEPDNINGKTC (SEQ ID NO: 311), GSAGSAAGSGEF (SEQ ID NO: 312),

SIVAQLSRPDPA (SEQ ID NO: 313), MKIIEQLPSA (SEQ ID NO: 314), VRHKLKRVGS (SEQ ID NO: 315), GHGTGSTGSGSS (SEQ ID NO: 316), MSRPDPA (SEQ ID NO: 317), GSAGSAAGSGEF (SEQ ID NO: 312), SGSETPGTSESA (SEQ ID NO: 318),

SGSETPGTSESATPEGGSGGS (SEQ ID NO: 319), or GGSM (SEQ ID NO: 320).

Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure.

[0241] In some embodiments, the nucleobase editor comprises a guide nucleotide sequence- programmable DNA-binding protein domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC 1) deaminase domain, where the deaminase domain is fused to the N- terminus of the napDNAbp domain via a linker comprising the amino acid sequence

SGSETPGTSESATPES (SEQ ID NO: 310). In some embodiments, the a guide nucleotide sequence-programmable DNA-binding protein domain comprises the amino acid sequence of any of the a guide nucleotide sequence-programmable DNA-binding protein domains provided herein. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 288). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 286). In some embodiments, the deaminase is pmCDAl (SEQ ID NO: 289). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 279). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 290-292). In some

embodiments, the fusion protein comprises a guide nucleotide sequence-programmable DNA-binding protein domain and an apolipoprotein B mRNA-editing complex 1 catalytic polypeptide-like 3G (APOBEC3G) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the a guide nucleotide sequence-programmable DNA-binding protein domain domain via a linker of any length or composition (e.g., an amino acid sequence, a peptide, a polymer, or a bond). In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 310). In some embodiments, the linker comprises the amino acid sequence (SGGS) ₂ SGSETPGTSESATPES(SGGS) ₂ (SEQ ID NO: 1069).

[0242] In some embodiments, the fusion protein comprises a guide nucleotide sequence- programmable DNA-binding protein domain and a cytidine deaminase 1 (CDA1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 310). In some embodiments, the linker comprises the amino acid sequence (SGGS) ₂ SGSETPGTSESATPES(SGGS) ₂ (SEQ ID NO: 1069). In some embodiments, the guide nucleotide sequence-programmable DNA- binding protein domain comprises the amino acid sequence of any of the guide nucleotide sequence-programmable DNA-binding protein domains provided herein.

[0243] In some embodiments, the fusion protein comprises a guide nucleotide sequence- programmable DNA-binding protein and an activation-induced cytidine deaminase (AID) deaminase domain, where the deaminase domain is fused to the N-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 310). In some embodiments, the linker comprises the amino acid sequence (SGGS) ₂ SGSETPGTSESATPES(SGGS) ₂ (SEQ ID NO: 1069). In some embodiments, the guide nucleotide sequence-programmable DNA- binding protein comprises the amino acid sequence of any of the guide nucleotide sequence- programmable DNA-binding protein domains provided herein.

[0244] Some aspects of the disclosure are based on the recognition that certain

configurations of a guide nucleotide sequence-programmable DNA-binding protein, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain fused to the N-terminus of a guide nucleotide sequence-programmable DNA-binding protein via a linker comprising the amino acid sequence

SGSETPGTSESATPES (SEQ ID NO: 310) was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. In some embodiments, the fusion protein comprises a guide nucleotide sequence-programmable DNA-binding protein domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC 1) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 310). In some embodiments, the fusion protein comprises a guide nucleotide sequence-programmable DNA-binding protein domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC 1) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp via a linker comprising the amino acid sequence SGGS) ₂ SGSETPGTSESATPES(SGGS) ₂ (SEQ ID NO: 1069).

[0245] To successfully edit the desired target C base, the linker between Cas9 and

APOBEC may be optimized, as described in Komor et ah, Nature, 533, 420-424 (2016), which is incorporated herein by reference. The numbering scheme for base editing is based on the predicted location of the target C within the single stranded stretch of DNA (R-loop) displaced by a programmable guide RNA sequence occurring when a DNA-binding domain (e.g. Cas9, nCas9, dCas9) binds a genomic site (see Figure 6). Conveniently, the sequence immediately surrounding the target C also matches the sequence of the guide RNA. The numbering scheme for base editing is based on a standard 20-mer programmable sequence, and defines position "21" as the first DNA base of the PAM sequence, resulting in position "1" assigned to the first DNA base matching the 5'-end of the 20-mer programmable guide RNA sequence. Therefore, for all Cas9 variants, position "21" is defined as the first base of the PAM sequence (e.g. NGG, NGAN, NGNG, NGAG, NGCG, NNGRRT, NGRRN, NNNRRT, NNNGATT, NNAGAA, NAAAC). When a longer programmable guide RNA sequence is used (e.g. 21-mer) the 5'-end bases are assigned a decreasing negative number starting at "-1". For other DNA-binding domains that differ in the position of the PAM sequence, or that require no PAM sequence, the programmable guide RNA sequence is used as a reference for numbering. A 3-aa linker gives a 2-5 base editing window (e.g. , positions 2, 3, 4, or 5 relative to the PAM sequence at position 21). A 9-aa linker gives a 3-6 base editing window (e.g. , positions 3, 4, 5, or 6 relative to the PAM sequence at position 21). A 16-aa linker (e.g., the SGSETPGTSESATPES (SEQ ID NO: 310) linker) gives a 4-7 base editing window (e.g. , positions 4, 5, 6, or 7 relative to the PAM sequence at position 21). A 21-aa linker gives a 5-8 base editing window (e.g., positions 5, 6, 7, 8 relative to the PAM sequence at position 21). Each of these windows can be useful for editing different targeted C bases. For example, the targeted C bases may be at different distances from the adjacent PAM sequence, and by varying the linker length, the precise editing of the desired C base is ensured. One skilled in the art, based on the teachings of CRISPR/Cas9 technology, in particular the teachings of US Provisional Applications 62/245828, 62/279346, 62/311,763, 62/322178, 62/357352, 62/370700, and 62/398490, and in Komor et al, Nature,

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), each of which is incorporated herein by reference, will be able to determine the window of editing for his/her purpose, and properly design the linker of the cytosine deaminase-dCas9 protein for the precise targeting of the desired C base.

[0246] To successfully edit the desired target C base, appropriate Cas9 domain may be selected to attached to the deaminase domain (e.g., APOBECl), since different Cas9 domains may lead to different editing windows, as described in in US Patent 9,068,179, US Patent Application Publications US 2015/0166980, US 2015/0166981, US 2015/0166982,

US20150166984, and US20150165054, and US Provisional Applications, U.S.S.N.

62/245,828, filed October 23, 2015; 62/279,346, filed January 15, 2016; 62/311,763, filed March 22, 2016; 62/322178, filed April 13, 2016, 62/357,352, filed June 30, 2016,

62,370,700, filed August 3, 2016; 62/398,490, filed September 22, 2016; 62/408686, filed October 14, 2016; PCT Application PCT/US2016/058344, filed October 22, 2016, US Patent Appliation 15/311,852, filed October 22, 2016; and in Komor et ah, Nature, Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage, 533, 420- 424 (2016), the entire contents of each of which are incorporated herein by reference. For example, APOBECl-XTEN-SaCas9n-UGI gives a 1-12 base editing window (e.g., positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 relative to the NNNRRT PAM sequence in positions 20- 26). One skilled in the art, based on the teachings of CRISPR/Cas9 technology, will be able to determine the editing window for his/her purpose, and properly determine the required Cas9 homolog and linker attached to the cytosine deaminase for the precise targeting of the desired C base.

[0247] In some embodiments, the fusion protein useful in the present disclosure further comprises a uracil glycosylase inhibitor (UGI) domain. A "uracil glycosylase inhibitor" refers to a protein that inhibits the activity of uracil-DNA glycosylase. The C to T base change induced by deamination results in a U:G heteroduplex, which triggers cellular DNA -repair response. Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells and initiates base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. Thus, such cellular DNA-repair response may be responsible for the decrease in nucleobase editing efficiency in cells. Uracil DNA Glycosylase Inhibitor (UGI) is known in the art to potently blocks human UDG activity. As described in Komor et ah, Nature (2016), fusing a UGI domain to the cytidine deaminase-dCas9 fusion protein reduced the activity of UDG and significantly enhanced editing efficiency.

[0248] Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et ah, Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264: 1163- 1171(1989); Lundquist et ah, Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997);

Ravishankar et ah, X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al, Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol. 287:331-346(1999), each of which is incorporated herein by reference. In some embodiments, the UGI domain comprises the amino acid sequence of SEQ ID NO: 304 without the N-terminal methionine (M). In some embodiments, the UGI comprises the following amino acid sequence:

Bacillus phage PBS2 (Bacteriophage PBS2)Uracil-DNA glycosylase inhibitor

MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWAL VIQDSNGENKIKML (SEQ ID NO: 304)

[0249] In some embodiments, the UGI protein comprises a wild type UGI or a UGI as set forth in SEQ ID NO: 304. In some embodiments, the UGI proteins useful in the present disclosure include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 304. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 304 or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 304. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as "UGI variants." A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to a wild type UGI or a UGI as set forth in SEQ ID NO: 304. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to the corresponding fragment of wild type UGI or a UGI as set forth in SEQ ID NO: 304.

[0250] It should be appreciated that additional proteins may be uracil glycosylase inhibitors. For example, other proteins that are capable of inhibiting (e.g., sterically blocking) a uracil- DNA glycosylase base-excision repair enzyme are within the scope of this disclosure. In some embodiments, a uracil glycosylase inhibitor is a protein that binds DNA. In some embodiments, a uracil glycosylase inhibitor is a protein that binds single-stranded DNA. For example, a uracil glycosylase inhibitor may be a Erwinia tasmaniensis single-stranded binding protein. In some embodiments, the single-stranded binding protein comprises the amino acid sequence (SEQ ID NO: 305). In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil. In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil in DNA. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein that does not excise uracil from the DNA. For example, a uracil glycosylase inhibitor is a UdgX. In some embodiments, the UdgX comprises the amino acid sequence (SEQ ID NO: 306). As another example, a uracil glycosylase inhibitor is a catalytically inactive UDG. In some

embodiments, a catalytically inactive UDG comprises the amino acid sequence (SEQ ID NO: 307). It should be appreciated that other uracil glycosylase inhibitors would be apparent to the skilled artisan and are within the scope of this disclosure.

[0251] Erwinia tasmaniensis SSB (themostable single- stranded DNA binding protein) MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETKEKTEW HRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTTEVVVNVGGT MQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGGAQQQARPQQQPQ QNNAPANNEPPIDFDDDIP (SEQ ID NO: 305)

[0252] UdgX (binds to Uracil in DNA but does not excise)

MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARIMMIGEQPG DKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTRAAGGKRRIHKT PSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGNDFRVTQHRGEVLHVDDV PGDPALVATVHPSSLLRGPKEERESAFAGLVDDLRVAADVRP (SEQ ID NO: 306)

[0253] UDG (catalytically inactive human UDG, binds to Uracil in DNA but does not excise)

MIGQKTLYS FFS PS P ARKRH APS PEP A VQGTG V AG VPEES GD A A AIP AKKAP AGQEE PGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKKHLSGEFGKPYFIKL MGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVILGQEPYHGPNQAHGLCFSV QRP VPPPPS LENIYKELS TDIEDFVHPGHGDLS GW AKQG VLLLN A VLT VRAHQ ANS H KERGWEQFTDAVVSWLNQNSNGLVFLLWGSYAQKKGSAIDRKRHHVLQTAHPSPL S VYRGFFGCRHFS KTNELLQKS GKKPIDWKEL (SEQ ID NO: 307)

[0254] In some embodiments, the UGI domain is fused to the C-terminus of the dCas9 domain in the fusion protein. Thus, the fusion protein would have an architecture of NH ₂ - [cytosine deaminase] -[guide nucleotide sequence-programmable DNA-binding protein domain]-[UGI]-COOH. In some embodiments, the UGI domain is fused to the N-terminus of the cytosine deaminase domain. As such, the fusion protein would have an architecture of NH ₂ -[UGI]-[cytosine deaminase] -[guide nucleotide sequence-programmable DNA-binding protein domain]-COOH. In some embodiments, the UGI domain is fused between the guide nucleotide sequence-programmable DNA-binding protein domain and the cytosine deaminase domain. As such, the fusion protein would have an architecture of NH ₂ -[cytosine deaminase]- [UGI]-[guide nucleotide sequence-programmable DNA-binding protein domain]-COOH. The linker sequences described herein may also be used for the fusion of the UGI domain to the cytosine deaminase-dCas9 fusion proteins.

[0255] In some embodiments, the fusion protein comprises the structure:

[cytosine deaminase] -[optional linker sequence] -[guide nucleotide sequence-programmable

DNA binding protein] -[optional linker sequence] -[UGI];

[cytosine deaminase] -[optional linker sequence] -[UGI] -[optional linker sequence] -[ guide nucleotide sequence-programmable DNA binding protein] ;

[UGI] -[optional linker sequence]-[cytosine deaminase] -[optional linker sequence] -[guide nucleotide sequence-programmable DNA binding protein] ;

[UGI] -[optional linker sequence] -[guide nucleotide sequence-programmable DNA binding protein] -[optional linker sequence] -[cytosine deaminase];

[guide nucleotide sequence-programmable DNA binding protein] -[optional linker sequence] - [cytosine deaminase] -[optional linker sequence] -[UGI]; or

[guide nucleotide sequence-programmable DNA binding protein] -[optional linker sequence] -

[UGI] -[optional linker sequence]-[cytosine deaminase].

[0256] In some embodiments, the fusion protein comprises the structure:

[cytosine deaminase] -[optional linker sequence] -[Cas9 nickase]- [optional linker sequence] -

[UGI];

[cytosine deaminase] -[optional linker sequence] -[UGI] -[optional linker sequence]-[Cas9 nickase] ;

[UGI] -[optional linker sequence]-[cytosine deaminase] -[optional linker sequence]-[Cas9 nickase] ;

[UGI] -[optional linker sequence]-[Cas9 nickase] -[optional linker sequence] -[cytosine deaminase] ;

[Cas9 nickase] -[optional linker sequence] -[cytosine deaminase] -[optional linker sequence]-

[UGI]; or

[Cas9 nickase] -[optional linker sequence] -[UGI] -[optional linker sequence] -[cytosine deaminase] .

[0257] In some embodiments, fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. In some embodiments, the NLS is fused to the N-terminus of the guide nucleotide sequence- programmable DNA-binding protein domain. In some embodiments, the NLS is fused to the C-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain. In some embodiments, the NLS is fused to the N-terminus of the cytosine deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. Non-limiting, exemplary NLS sequences may be PKKKRKV (SEQ ID NO: 829) or

MDS LLMNRRKFLYQFKNVRW AKGRRET YLC (SEQ ID NO: 830).

[0258] In some embodiments, any of the fusion proteins provided herein comprise a second UGI domain. Fusion proteins comprising two UGI domains are described in U.S.

Provisional Applications, U.S.S.N. 62/475,830, filed March 23, 2017; 62/490,587;

62/511,934, filed May 26, 2017; 62/551/951, filed August 30, 2017; and Komor et al. (2017) Improved Base Excision Repair Inhibition and Bateriophage Mu Gam Protein Yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 3: eaao4774; the entire contents of which is incorporated herein by reference. In some embodiments, the second UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 304. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, the second UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 304. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 304. In some embodiments, the second UGI domain comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 304 or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 304. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as "UGI variants." A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 304. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild- type UGI or a UGI as set forth in SEQ ID NO: 304.

[0259] In some embodiments, the fusion protein comprises the structure:

[deaminase] -[optional linker sequence]-[dCas9]-[optional linker sequence] -[first

UGI] -[optional linker sequence]-[second UGI];

[deaminase] -[optional linker sequence]-[Cas9 nickase]- [optional linker sequence]- [first UGI] -[optional linker sequence] -[second UGI]; or

[deaminase] -[optional linker sequence]-[Cas9]-[optional linker sequence] -[first UGI]-

[optional linker sequence] -[second UGI].

[0260] In some embodiments, the nucleobase editor comprises a guide nucleotide sequence- programmable DNA-binding protein domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N- terminus of the guide nucleotide sequence-programmable DNA-binding protein domain via a linker comprising the amino acid sequence (SGGS) ₂ SGSETPGTSESATPES(SGGS) ₂ (SEQ ID NO: 1069). In some embodiments, the a guide nucleotide sequence-programmable DNA- binding protein domain comprises the amino acid sequence of any of the a guide nucleotide sequence-programmable DNA-binding protein domains provided herein. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 288). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 286). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 290-292). In some embodiments, the nucleobase editor comprises a first UGI domain fused to the C-terminus of a guide nucleotide sequence-programmable DNA-binding protein domain via a linker comprising the amino acid sequence (GGS) _n (SEQ ID NO: 784), wherein n is 3. In some embodiments, the nucleobase editor comprises a second UGI domain fused to the C-terminus of a first UGI domain via a linker comprising the amino acid sequence (GGS) _n (SEQ ID NO: 784), wherein n is 3.

[0261] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1084. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 1084.

[0262] In some embodiments, any of the fusion proteins provided herein may further comprise a Gam protein. The term "Gam protein," as used herein, refers generally to proteins capable of binding to one or more ends of a double strand break of a double stranded nucleic acid (e.g., double stranded DNA). In some embodiments, the Gam protein prevents or inhibits degradation of one or more strands of a nucleic acid at the site of the double strand break. In some embodiments, a Gam protein is a naturally-occurring Gam protein from bacteriophage Mu, or a non-naturally occurring variant thereof. Fusion proteins comprising Gam proteins are described in Komor et al. (2017) Improved Base Excision Repair Inhibition and Bateriophage Mu Gam Protein Yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 3: eaao4774; the entire contents of which is incorporated by reference herein. In some embodiments, the Gam protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence provided by SEQ ID NO: 3027. In some embodiments, the Gam protein comprises the amino acid sequence of SEQ ID NO: 3027. In some embodiments, the fusion protein (e.g., BE4-Gam of SEQ ID NO: 3028) comprises a Gam protein, wherein the Cas9 domain of BE4 is replaced with any of the Cas9 domains provided herein.

[0263] Gam from bacteriophage Mu:

AKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIAP IKTDIETLSKGVQGW CEANRDELTNGGKVKTANLVTGDVSWRVRPPSVS IRGMDAVMETLERLGLQRFIRTKQEINKEAILLEPKAVAGV

AGITVKSGIEDFS I IPFEQEAGI (SEQ ID NO: 1070)

[0264] BE4-Gam:

MAKPAKRIKSAAAAYVPQNRDAVITDIKRIGDLQREASRLETEMNDAIAEITEKFAARIA PIKTDIETLSKGVQG WCEANRDELTNGGKVKTANLVTGDVSWRVRPPSVSIRGMDAYMETLERLGLQRFIRTKQE INKEAILLEPKAVAG VAGITVKSGIEDFSIIPFEQEAGISGSETPGTSESATPESSSETGPVAVDPTLRRRIEPH EFEVFFDPRELRKET CLLYEINWGGRHS IWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCS ITWFLSWSPCGECSRAITEFLSRYPHVT LFIYIARLYHHADPRNRQGLRDLI SSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCI I LGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTS ESATPESSGGSSGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADL RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLI AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YIDGGASQEEFYKFI KPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKI I KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR LSRKLINGIRDKQSG KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKWDELVKVM GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLT KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVR EINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF FYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLI IKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEI IEQI SEF SKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDI IEKETGKQLVIQES ILMLPEEVEEVIGNKPESDILVHTAYDE STDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDI IEKETGKQLVIQES ILMLPEEVEE

VIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS PKKKRK (SEQ ID NO:

1071)

[0265] Some aspects of the present disclosure provide nucleobase editors associated with a guide nucleotide sequence (e.g., a guide RNA or gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though "gRNA" is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as a single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), which is incorporated herein by reference. Other examples of gRNAs {e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S. S.N. 61/874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases And Uses Thereof," and U.S. Provisional Patent Application, U.S.S.N. 61/874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," each of which isincorporated herein by reference in their entirety. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. These proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage {e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Science 339, 819-823 (2013); Mali, P. et al. Science 339, 823-826 (2013); Hwang, W.Y. et al. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. eLife 2, e00471 (2013); Dicarlo, J.E. et al. Nucleic acids research (2013); Jiang, W. et al. Nature Biotechnology 31, 233-239 (2013); each of which are incorporated herein by reference). In particular, examples of guide nucleotide sequences {e.g., sgRNAs) that may be used to target the fusion protein of the present disclosure to its target sequence to deaminate the targeted C bases are described in Komor et al., Nature, 533, 420-424 (2016), which is incorporated herein by reference.

[0266] The specific structure of the guide nucleotide sequences {e.g., sgRNAs) depends on its target sequence and the relative distance of a PAM sequence downstream of the target sequence. One skilled in the art will understand, that no unifying structure of guide nucleotide sequence is given, for that he target sequences are different for each and every C targeted to be deaminated.

[0267] However, the present disclosure provides guidance in how to design the guide nucleotide sequence, e.g., an sgRNA, so that one skilled in the art may use such teaching to a target sequence of interest. An gRNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to fusion proteins disclosed herein. In some embodiments, the guide RNA comprises a structure 5 '-[guide sequence] -tracrRNA-3'. Non-limiting, exemplary tracrRNA sequences are shown in Table 11. Table 11. TracrRNA orthologues and sequences

[0268] The guide sequence of the gRNA comprises a sequence that is complementary to the target sequence. The guide sequence is typically about 20 nucleotides long. For example, the guide sequence may be 15-25 nucleotides long. In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some

embodiments, the guide sequence is more than 25 nucleotides long. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited.

[0269] In some embodiments, the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence.

Compositions

[0270] Some aspects of the present disclosure relate to compositions that may be used for generating cancer vaccines in vivo or ex vivo. In some embodiments, the composition comprises: (i) a nucleobase editor or a nucleic acid molecule encoding the nucleobase editor described herein; and (ii) a guide nucleotide sequence targeting the nucleobase editor to a tumor specific antigen-encoding polynucleotide. The guide nucleotide sequence that may be used to generate heteroclitic or cryptic epitopes may be selected from SEQ ID NOs: X-X. Guide nucleotide sequences for generating specific heteroclitic or cryptic epitopes may be found in Tables 5 and 6.

[0271] In some embodiments, the composition described herein further comprises a pharmaceutically acceptable carrier.

[0272] As used here, the term "pharmaceutically-acceptable carrier" means a

pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the

formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

[0273] In some embodiments, the nucleobase editors and the guide nucleotides of the present disclosure in a composition is administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non- porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. In some embodiments, the injection is directed to the liver.

[0274] In other embodiments, the nucleobase editors and the guide nucleotides are delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228: 190; During et al, 1989, Ann. Neurol. 25:351;

Howard et al., 1989, J. Neurosurg. 71: 105.) Other controlled release systems are discussed, for example, in Langer, supra. [0275] In typical embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human . Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0276] A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

[0277] The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et ah, Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethyls ulfate, or "DOTAP," are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951;

4,920,016; and 4,921,757.

[0278] The pharmaceutical compositions of this disclosure may be administered or packaged as a unit dose, for example. The term "unit dose" when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle. [0279] In some embodiments, the nucleobase editors or the guide nucleotides described herein may be conjugated to a therapeutic moiety, e.g., an anti-inflammatory agent.

Techniques for conjugating such therapeutic moieties to polypeptides, including e.g., Fc domains, are well known; see, e.g., Amon et al., "Monoclonal Antibodies For

Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62: 119-158.

[0280] Further, the compositions of the present disclosure may be assembled into kits. In some embodiments, the kit comprises nucleic acid vectors for the expression of the nucleobase editors described herein. In some embodiments, the kit further comprises appropriate guide nucleotide sequences {e.g., gRNAs) or nucleic acid vectors for the expression of such guide nucleotide sequences, to target the nucleobase editors to the desired target sequences.

[0281] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions of uses. Any of the kit described herein may further comprise components needed for performing the assay methods. Each component of the kits, where applicable, may be provided in liquid form {e.g., in solution), or in solid form, {e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible {e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or certain organic solvents), which may or may not be provided with the kit.

[0282] In some embodiments, the kits may optionally include instructions and/or promotion for use of the components provided. As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the

instructions are to be associated with the kit, for example, audiovisual {e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, "promoted" includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0283] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively the kits may include the active agents premixed and shipped in a vial, tube, or other container.

[0284] The kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat

sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.

Therapeutics

[0285] The compositions or cancer vaccines (e.g., a whole-cell vaccine comprising a modified tumor cell) described herein may be administered to a subject in need thereof, in a therapeutically effective amount, to treat cancer. The compositions and cancer vaccines described herein induce tumor- specific adaptive responses. It is known that cancer cells exploit immune checkpoints to evade immune surveillance. Thus, in some embodiments, in addition to the compositions or cancer vaccines described herein, agents that modulate the activities of immune checkpoints are also administered to boost the tumor- specific immune response elicited by the cancer vaccines.

[0286] In some embodiments, the agents that modulate the activities of immune

checkpoints are immune checkpoint inhibitors. "Immune checkpoints" are proteins in the immune system that either enhance an immune response signal (co- stimulatory molecules) or reduce an immune response signal. Many cancers protect themselves from the immune system by exploiting the inhibitory immune checkpoint proteins to inhibit the T cell signal. Such inhibitory checkpoint proteins include, without limitation, Cytotoxic T-Lymphocyte- Associated protein 4 (CTLA-4), Programmed Death 1 receptor (PD-1), T-cell

Immunoglobulin domain and Mucin domain 3 (TIM3), Lymphocyte Activation Gene-3 (LAG3), V-set domain-containing T-cell activation inhibitor 1 (VTVN1 or B7-H4), Cluster of Differentiation 276 (CD276 or B7-H3), B and T Lymphocyte Attenuator (BTLA), Galectin-9 (GAL9), Checkpoint kinase 1 (Chkl), Adenosine A2A receptor (A2aR),

Indoleamine 2,3-dioxygenase (IDO), Killer-cell Immunoglobulin-like Receptor (KIR), Lymphocyte Activation Gene-3 (LAG3), and V-domain Ig suppressor of T cell activation (VISTA).

[0287] Some of these immune checkpoint proteins need their cognate binding partners, or ligands, for their immune inhibitory activity. For example, A2AR is the receptor of adenosine A2A and binding of A2A to A2AR activates a negative immune feedback loop. As another example, PD-1 associates with its two ligands, PD-L1 and PD-L2, to down regulate the immune system by preventing the activation of T-cells. PD-1 promotes the programmed cell death of antigen specific T-cells in lymph nodes and simultaneously reduces programmed cell death of suppressor T cells, thus achieving its immune inhibitory function. As yet another example, CTLA4 is present on the surface of T cells, and when bound to its binding partner CD80 or CD86 on the surface of antigen-present cells (APCs), it transmits an inhibitory signal to T cells, thereby reducing the immune response.

[0288] Cancer cells are known to exploit the immune checkpoint proteins to escape being attacked by the immune system. Therefore, the use of immune checkpoint inhibitors to enhance an immune response against cancer, and thus treating cancer, have been described. The immunotherapeutic agents in the compositions of the present disclosure may also be immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibits any one or more of Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), Programmed Death 1 receptor (PD-1), T-cell Immunoglobulin domain and Mucin domain 3 (TIM3), Lymphocyte Activation Gene-3 (LAG3), V-set domain-containing T-cell activation inhibitor 1 (VTVN1 or B7-H4), Cluster of Differentiation 276 (CD276 or B7-H3), B and T Lymphocyte Attenuator (BTLA), Galectin-9 (GAL9), Checkpoint kinase 1 (Chkl), Adenosine A2A receptor (A2aR), Indoleamine 2,3-dioxygenase (IDO), Killer-cell Immunoglobulin-like Receptor (KIR), Lymphocyte Activation Gene-3 (LAG3) and V-domain Ig suppressor of T cell activation (VISTA).

[0289] An "immune checkpoint inhibitor" is a molecule that prevents or weakens the activity of an immune checkpoint inhibitor, For example, an immune checkpoint inhibitor may inhibit the binding of the immune checkpoint protein to its cognate binding partner, e.g. , PD-1, CTLA-4, or A2aR. In some embodiments, the immune checkpoint inhibitor is a small molecule. In some embodiments, the immune checkpoint inhibitors is a nucleic acid aptamer (e.g., a siRNA targeting any one of the immune checkpoint proteins). In some embodiments, the immune checkpoint inhibitor is a recombinant protein. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the antibody comprises an anti- CTLA-4, anti-PD- 1, anti-PD-Ll, anti-TIM3, anti-LAG3, anti-B7-H3, anti-B7-H4, anti- BTLA, anti-GAL9, anti-Chk, anti-A2aR, anti-IDO, anti-KIR, anti-LAG3, anti- VISTA antibody, or a combination of any two or more of the foregoing antibodies. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody. In some embodiments, the immune checkpoint inhibitor comprises anti-PDl, anti-PD-Ll, anti- CTLA-4, or a combination of any two or more of the foregoing antibodies. For example, the anti-PD- 1 antibody is pembrolizumab (Keytruda ^® ) or nivolumab (Opdivo ^® ) and the anti- CTLA-4 antibody is ipilimumab (Yervoy ^® ). Thus, in some embodiments, the immune checkpoint inhibitor comprises pembrolizumab, nivolumab, ipilimumab, or any combination of two or more of the foregoing antibodies. The examples described herein are not meant to be limiting and that any immune checkpoint inhibitors known in the art and any combinations thereof may be used in accordance with the present disclosure.

[0290] In some embodiments, the immune checkpoint may be inhibited by disrupting any one of the immune checkpoint genes (e.g., CTLA-4, PD-1, PD-L1, TIM3, LAG3, B7-H3, B7- H4, BTLA, GAL9, Chkl , or A2aR) using any of the gene editing methods known in the art (e.g., CRISPR/Cas9 mediated cleavage of any of the genes).

[0291] In some embodiments, an adjuvant is futher administered to the subject. Adjuvants are substances which enhance the immune response when administered together with an immunogen or antigen. Adjuvants are thought to function in several ways, including by increasing the surface area of antigen, prolonging the retention of the antigen in the body thus allowing time for the lymphoid system to have access to the antigen, slowing the release of antigen, targeting antigen to macrophages, activating macrophages, or otherwise eliciting non-specific activation of the cells of the immune system see, e.g., H. S. Warren et al, Annu. Rev. immunol., 4:369 (1986). Currently, an essential role of adjuvants in vaccines is to direct CD4+ T cell subset differentiation, although how adjuvants perform this function is poorly understood.

[0292] The ability of a adjuvant to induce and increase a specific type of immune response and the identification of that ability is thus a key factor in the selection of particular adjuvants for vaccine use against a particular pathogen. Typical adjuvants include water and oil emulsions, e.g. , Freund' s adjuvant, and chemical compounds such as aluminum hydroxide or alum. At present, alum is the only adjuvant approved in the United States for human vaccines; it has been determined that alum induces the production of TH 2 cells.

[0293] Many of the most effective adjuvants include bacteria or their products, e.g., microorganisms such as the attenuated strain of Mycobacterium bovis, bacillus Calmette- Guerin (BCG); microorganism components, e.g., alum-precipitated diphtheria toxoid, bacterial lipopoly saccharide and endotoxins. However, the role that the bacteria play is ill- defined. Recently, it has been noted that many bacteria or their products, lipopolysaccharide, Staphylococcus aureus, Mycobacterium tuberculosis, and C. parvum, stimulate IL- 12 production by macrophages A. D'Andrea et al, J. Exp. Med., 176: 1387 (1992).

[0294] Cancers or tumors include but are not limited to neoplasms, malignant tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer may be a primary or metastatic cancer. Cancers include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS- associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen' s disease and Paget' s disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi' s sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non- seminoma, teratomas, choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer.

[0295] "A therapeutically effective amount" as used herein refers to the amount of each base-editing agent of the present disclosure required to confer therapeutic effect on the subject, either alone or in combination with one or more other therapeutic agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual subject parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, therapeutic agents that are compatible with the human immune system, such as polypeptides comprising regions from humanized antibodies or fully human antibodies, may be used to prolong the half-life of the polypeptide.

[0296] Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a disease. Alternatively, sustained continuous release formulations of a polypeptide or a polynucleotide may be appropriate. Various formulations and devices for achieving sustained release are known in the art. In some embodiments, dosage is daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays.

[0297] The dosing regimen (including the polypeptide used) can vary over time. In some embodiments, for an adult subject of normal weight, doses ranging from about 0.01 to 1000 mg/kg may be administered. In some embodiments, the dose is between 1 to 200 mg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular subject and that subject's medical history, as well as the properties of the polypeptide or the polynucleotide (such as the half-life of the polypeptide or the polynucleotide, and other considerations well known in the art).

[0298] For the purpose of the present disclosure, the appropriate dosage of a therapeutic agent as described herein will depend on the specific agent (or compositions thereof) employed, the formulation and route of administration, the type and severity of the disease, whether the polypeptide or the polynucleotide is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the antagonist, and the discretion of the attending physician. Typically the clinician will administer a

polypeptide until a dosage is reached that achieves the desired result.

[0299] Administration of one or more agents can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an agent (e.g., cancer vaccine) may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g. , either before, during, or after developing a disease. "Treat," as used herein, means to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease. "Treating," as used herein refers to the application or administration of a composition or a cancer vaccine described herein to a subject in need thereof.

[0300] "A subject in need thereof, refers to an individual who has a disease, a symptom of the disease, or a predisposition toward the disease. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is human. Alleviating a disease includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results.

[0301] As used therein, "delaying" the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0302] "Development" or "progression" of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset.

[0303] As used herein "onset" or "occurrence" of a disease includes initial onset and/or recurrence. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g. , administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

[0304] The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0305] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

[0306] In order that the disclosure described herein may be more fully understood, the following examples are set forth. The synthetic examples described in this application are offered to illustrate the compounds and methods provided herein and are not to be construed in any way as limiting their scope. Example 1: Guide nucleotide sequence-programmable DNA-binding protein domains, deaminases, and base editors

[0307] Non-limiting examples of suitable guide nucleotide sequence-programmable DNA- binding protein domain s are provided. The disclosure provides Cas9 variants, for example, Cas9 proteins from one or more organisms, which may comprise one or more mutations (e.g., to generate dCas9 or Cas9 nickase). In some embodiments, one or more of the amino acid residues, identified below by an asterek, of a Cas9 protein may be mutated. In some embodiments, the D10 and/or H840 residues of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, are mutated. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to any amino acid residue, except for D. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to an A. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is an H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to any amino acid residue, except for H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to an A. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is a D.

[0308] A number of Cas9 sequences from various species were aligned to determine whether corresponding homologous amino acid residues of D10 and H840 of SEQ ID NO: 1 or SEQ ID NO: 11 can be identified in other Cas9 proteins, allowing the generation of Cas9 variants with corresponding mutations of the homologous amino acid residues. The alignment was carried out using the NCBI Constraint-based Multiple Alignment Tool (COBALT(accessible at st-va.ncbi. nlm.nih.gov/tools/cobalt), with the following parameters. Alignment parameters: Gap penalties -11,-1; End-Gap penalties -5,-1. CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on. Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8;

Alphabet Regular. [0309] An exemplary alignment of four Cas9 sequences is provided below. The Cas9 sequences in the alignment are: Sequence 1 (S I): SEQ ID NO: 11 I WP_010922251I gi 499224711 I type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus pyogenes]; Sequence 2 (S2): SEQ ID NO: 12 I WP_039695303 I gi 746743737 I type II CRISPR RNA- guided endonuclease Cas9 [Streptococcus gallolyticus]; Sequence 3 (S3): SEQ ID NO: 13 I WP_045635197 I gi 782887988 I type II CRISPR RNA-guided endonuclease Cas9

[Streptococcus mitis]; Sequence 4 (S4): SEQ ID NO: 14 I 5AXW_A I gi 924443546 I Staphylococcus Aureus Cas9. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. Amino acid residues 10 and 840 in S I and the homologous amino acids in the aligned sequences are identified with an asterisk following the respective amino acid residue.

51 1 —MDKK-YSIGLD*IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI—GALL FDSG—ETAEATRLKRTARRRYT 73

52 1 —MTKKNYSIGLD*IGTNSVGWAVITDDYKVPAKKMKVLGNTDKKYIKKNLL—GALL FDSG—ETAEATRLKRTARRRYT 74

53 1 —M-KKGYSIGLD*IGTNSVGFAVITDDYKVPSKKMKVLGNTDKRFIKKNLI—GALL FDEG—TTAEARRLKRTARRRYT 73

54 1 GSHMKRNYILGLD*IGITSVGYGII—DYET RDVIDAGVRLFKEANVENNEGRRSKRGARRLKR 61

51 74 RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYP TIYHLRKKLVDSTDKADLRL 153

52 75 RRKNRLRYLQEIFANEIAKVDESFFQRLDESFLTDDDKTFDSHPIFGNKAEEDAYHQKFP TIYHLRKHLADSSEKADLRL 154

53 74 RRKNRLRYLQEIFSEEMSKVDSSFFHRLDDSFLIPEDKRESKYPIFATLTEEKEYHKQFP TIYHLRKQLADSKEKTDLRL 153

54 62 RRRHRIQRVKKLL FDYNLLTD HSELSGINPYEARVKGLSQKLSEEE 107

51 154 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEK 233

52 155 VYLALAHMIKFRGHFLIEGELNAENTDVQKIFADFVGVYNRTFDDSHLSEITVDVASILT EKISKSRRLENLIKYYPTEK 234

53 154 IYLALAHMIKYRGHFLYEEAFDIKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFT DKISKSAKRERVLKLFPDEK 233

54 108 FSAALLHLAKRRG VHNVNEVEEDT 131

SI 234 KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAA KNLSDAILLSDILRVNTEIT 313

S2 235 KNTLFGNLIALALGLQPNFKTNFKLSEDAKLQFSKDTYEEDLEELLGKIGDDYADLFTSA KNLYDAILLSGILTVDDNST 314

S3 234 STGLFSEFLKLIVGNQADFKKHFDLEDKAPLQFSKDTYDEDLENLLGQIGDDFTDLFVSA KKLYDAILLSGILTVTDPST 313

S4 132 GNELS TKEQISRN 144

51 314 KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY KFIKPILEKM—DGTEELLV 391

52 315 KAPLSASMIKRYVEHHEDLEKLKEFIKANKSELYHDIFKDKNKNGYAGYIENGVKQDEFY KYLKNILSKIKIDGSDYFLD 394

53 314 KAPLSASMIERYENHQNDLAALKQFIKNNLPEKYDEVFSDQSKDGYAGYIDGKTTQETFY KYIKNLLSKF—EGTDYFLD 391

54 145 SKALEEKYVAELQ LERLKKDG 165

51 392 KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEE 471

52 395 KIEREDFLRKQRTFDNGSIPHQIHLQEMHAILRRQGDYYPFLKEKQDRIEKILTFRIPYY VGPLVRKDSRFAWAEYRSDE 474

53 392 KIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEYYPFLKDNKEKIEKILTFRIPYY VGPLARGNRDFAWLTRNSDE 471

54 166 —EVRGSINRFKTSD YVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGP—GEGSPFGW K 227

SI 472 TITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLSGEQKKAIVDL 551

S2 475 KITPWNFDKVIDKEKSAEKFITRMTLNDLYLPEEKVLPKHSHVYETYAVYNELTKIKYVN EQGKE-SFFDSNMKQEIFDH 553

S3 472 AIRPWNFEEIVDKASSAEDFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKVKFIA EGLRDYQFLDSGQKKQIVNQ 551

IQKAQVSGQG|DSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMA |RENQTT

IQKSQWGDV|DDIEAWHDLPGSPAIKKGILQSVKIVDELVKVMG-GNPDNIVIEMA^ RENQTT

IQKAQVIGKT|DDVKQWQELSGSPAIKKGILQSIKIVDELVKVMG-HAPESIVIEMA |RENQTT

-KKVDLSQQK|EIPTTLVDDFILSPWKRSFIQSIKVINAIIKKYG—LPNDIIIEL A|REKNSKDAQKMINEMQKRNRQTN

TQRKFDN-LTKAERG|GL KAGFIKRQLVl - KAGFIKRQLVl - KVGFIKRQLVl |ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAWGTALIKKYP| |ETRQITKHVAQILDARFNTEHDENDKVIRDVKVITLKSNLVSQFRKDFEFYKVREINDY HHAHDAYLNAWGTALLKKYP| |ETRQITKHVAQILDARYNTEVNEKDKKNRTVKIITLKSNLVSNFRKEFRLYKVREINDY HHAHDAYLNAWAKAILKKYP| IDTRYATRGLMNLLRSYFRVN- -NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIA-

— RDFATVRKVLSMPQVNIVKKTEVQT|GGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLWAKV IDFEKVRKVLSYPQVNIVKKVETQT|GGFSKESILPKGDSDKLIPRKTKKVYWDTKKYGG FDSPTVAYSVFWADV KDFAIIKKVLSLPQVNIVKKREVQT|GGFSKESILPKGNSDKLIPRKTKDILLDTTKYGG FDSPVIAYSILLIADIHIKDFKDYKYSHRVDKKPNRELINDTLYST|RKDDKGNTLIVNN LNGLYDKDNDKL KKLIN-KSP EKLLMYHH EKGKSKKLKSVKELLGITIMERSSFEKNPI-DFLEAKG YKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKG EKGKAKKLKTVKELVGISIMERSFFEENPV-EFLENKG YHNIREDKLIKLPKYSLFEFEGGRRRLLASASELQKG EKGKAKKLKTVKTLVGITIMEKAAFEENPI-TFLENKG YHNVRKENILCLPKYSLFELENGRRRLLASAKELQKG DPQTYQKLK LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR NKV NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKH NEMVLPGYLVELLYHAHRADNF NSTEYLNYVSEHKKEFEKVLSCVEDFANLYVDVEKNLSKIRAVADSM S3 1231 NEIVLPVYLTTLLYHSKNVHKL DEPGHLEYIQKHRNEFKDLLNLVSEFSQKYVLADANLEKIKSLYADN 1299

S4 908 VKLSLKPYRFD-VYLDNGVYKFV TVKNLDVIK—KENYYEVNSKAYEEAKKLKKISNQAEFIASFYNNDLIKING 979

SI 1298 RDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT GLYETRI DLSQL 1365

S2 1302 DNFSIEEISNSFINLLTLTALGAPADFNFLGEKIPRKRYTSTKECLNATLIHQSIT GLYETRI DLSKL 1369

S3 1300 EQADIEILANSFINLLTFTALGAPAAFKFFGKDIDRKRYTTVSEILNATLIHQSIT GLYETWI DLSKL 1367

S4 980 ELYRVIGVNNDLLNRIEVNMIDITYR-EYLENMNDKRPPRI IKTIASKT QSIKKYSTDILGNLYEVKSKKHPQI IKK 1055

SI 1366 GGD 1368

S2 1370 GEE 1372

S3 1368 GED 1370

S4 1056 G— 1056

[0310] The alignment demonstrates that amino acid sequences and amino acid residues that are homologous to a reference Cas9 amino acid sequence or amino acid residue can be identified across Cas9 sequence variants, including, but not limited to Cas9 sequences from different species, by identifying the amino acid sequence or residue that aligns with the reference sequence or the reference residue using alignment programs and algorithms known in the art. This disclosure provides Cas9 variants in which one or more of the amino acid residues identified by an asterisk in SEQ ID NOs: 11- 14 (e.g., S I, S2, S3, and S4,

respectively) are mutated as described herein. The residues D10 and H840 in Cas9 of SEQ ID NO: 1 that correspond to the residues identified in SEQ ID NOs: 11- 14 by an asterisk are referred to herein as "homologous" or "corresponding" residues. Such homologous residues can be identified by sequence alignment, e.g. , as described above, and by identifying the sequence or residue that aligns with the reference sequence or residue. Similarly, mutations in Cas9 sequences that correspond to mutations identified in SEQ ID NO: 1 herein, e.g. , mutations of residues 10, and 840 in SEQ ID NO: 1, are referred to herein as "homologous" or "corresponding" mutations. For example, the mutations corresponding to the D10A mutation in SEQ ID NO: 1 or S I (SEQ ID NO: 11) for the four aligned sequences above are Dl 1A for S2, D10A for S3, and D13A for S4; the corresponding mutations for H840A in SEQ ID NO: 1 or S I (SEQ ID NO: 11) are H850A for S2, H842A for S3, and H560A for S4.

[0311] A total of 250 Cas9 sequences (SEQ ID NOs: 11-260) from different species are provided. Amino acid residues homologous to residues 10, and 840 of SEQ ID NO: 1 may be identified in the same manner as outlined above. All of these Cas9 sequences may be used in accordance with the present disclosure. WP_010922251.1 type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 11 WP_039695303.1 type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus gallolyticus ] SEQ ID NO WP_045635197.1 type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus mitis] SEQ ID NO 13 5AXW_A Cas9, Chain A, Crystal Structure [ Staphylococc is Aureus] SEQ ID NO: 14

WP_ _. 009880683. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 15

WP_ _. 010922251. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 16

WP_ _. 011054416. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 17

WP_ _. 011284745. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 18

WP_ _. 011285506. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 19

WP_ _. 011527619. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 20

WP_ _. 012560673. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 21

WP_ _. 014407541. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 22

WP_ _. 020905136. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 23

WP_ _. 023080005. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 24

WP_ _. 023610282. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 25

WP_ _. 030125963. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 26

WP_ _. 030126706. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 27

WP_ _. 031488318. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 28

WP_ _. 032460140. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 29

WP_ _. 032461047. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 30

WP_ _. 032462016. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 31

WP_ _. 032462936. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 32

WP_ _. 032464890. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 33

WP_ _. 033888930. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 34

WP_ _. 038431314. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 35

WP_ _. 038432938. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus pyogenes ] SEQ ID NO: 36

WP_038434062.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus pyogenes] SEQ ID NO: 37 BAQ51233.1 CRISPR-associated protein, Csnl family [Streptococcus pyogenes] SEQ ID NO: 38

KGE60162.1 hypothetical protein MGAS2111_0903 [Streptococcus pyogenes MGAS2111] SEQ ID NO: 39

KGE60856.1 CRISPR-associated endonuclease protein [Streptococcus pyogenes SS1447] SEQ ID NO: 40

WP_ _. 002989955 1 MULTISPECIES : type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus ] SEQ ID NO:

WP_ _. 003030002 1 MULTISPECIES : type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus ] SEQ ID NO:

WP_ _. 003065552 1 MULTISPECIES: type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus ] SEQ ID NO:

WP_ _. 001040076 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 44

WP_ _. 001040078 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 45

WP_ _. 001040080 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 46

WP_ _. 001040081 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 47

WP_ _. 001040083 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 48

WP_ _. 001040085 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 49

WP_ _. 001040087 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 50

WP_ _. 001040088 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 51

WP_ _. 001040089 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 52

WP_ _. 001040090 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 53

WP_ _. 001040091 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 54

WP_ _. 001040092 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 55

WP_ _. 001040094 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 56

WP_ _. 001040095 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 57

WP_ _. 001040096 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 58

WP_ _. 001040097 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 59

WP_ _. 001040098 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 60

WP_ _. 001040099 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 61

WP_ _. 001040100 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus agalactiae ] SEQ ID NO 62

WP_001040104.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 63 WP_001040105.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 64 WP_001040106.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 65 WP_001040107.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 66 WP_001040108.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 67 WP_001040109.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 68 WP_001040110.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 69 WP_015058523.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 70 WP_017643650.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 71 WP_017647151.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 72 WP_017648376.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 73 WP_017649527.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 74 WP_017771611.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 75 WP_017771984.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 76 CFQ25032.1 CRISPR-associated protein [Streptococcus agalactiae] SEQ ID NO: 77

CFV16040.1 CRISPR-associated protein [Streptococcus agalactiae] SEQ ID NO: 78

KLJ37842.1 CRISPR-associated protein Csnl [Streptococcus agalactiae] SEQ ID NO 79

KLJ72361.1 CRISPR-associated protein Csnl [Streptococcus agalactiae] SEQ ID NO 80

KLL20707.1 CRISPR-associated protein Csnl [Streptococcus agalactiae] SEQ ID NO 81

KLL42645.1 CRISPR-associated protein Csnl [Streptococcus agalactiae] SEQ ID NO 82

WP_047207273.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 83 WP_047209694.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 84 WP_050198062.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 85 WP_050201642.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 86 WP_050204027.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 87 WP_050881965.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae SEQ ID NO: 88

WP_050886065.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus agalactiae] SEQ ID NO: 89 AHN30376.1 CRISPR-associated protein Csnl [Streptococcus agalactiae 138P] SEQ ID NO: 90

EA078426.1 reticulocyte binding protein [Streptococcus agalactiae H36B] SEQ ID NO: 91

CCW42055.1 CRISPR-associated protein, SAG0894 family [Streptococcus agalactiae ILRI112] SEQ ID NO: 92

WP_003041502.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus anginosus] SEQ ID NO: 93 WP_037593752.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus anginosus] SEQ ID NO: 94 WP_049516684.1 CRISPR-associated protein Csnl [Streptococcus anginosus] SEQ ID NO: 95

GAD46167.1 hypothetical protein ANG6_0662 [Streptococcus anginosus T5] SEQ ID NO: 96

WP_ _. 018363470 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus caballi] SEQ ID NO: 97

WP_ _. 003043819 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus canis] SEQ ID NO: 98

WP_ _. 006269658 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus constellatus ] SEQ ID NO: 99

WP_ _. 048800889 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus constellatus ] SEQ ID NO: 100

WP_ _. 012767106 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus dysgalactiae ] SEQ ID NO: 101

WP_ _. 014612333 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus dysgalactiae ] SEQ ID NO: 102

WP_ _. 015017095 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus dysgalactiae ] SEQ ID NO: 103

WP_ _. 015057649 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus dysgalactiae ] SEQ ID NO: 104

WP_ _. 048327215 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus dysgalactiae ] SEQ ID NO: 105

WP_ _. 049519324 1 CRISPR-associated protein Csnl [Streptococcus dysgalactiae SEQ ID NO: 106

WP_ _. 012515931 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus equi] SEQ ID NO: 107

WP_ _. 021320964 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus equi] SEQ ID NO: 108

WP_ _. 037581760 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus equi] SEQ ID NO: 109

WP_ _. 004232481 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus equinus] SEQ ID NO: 110

WP_ _. 009854540 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus gallolyticus ] SEQ ID NO: 111

WP_ _. 012962174 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus gallolyticus ] SEQ ID NO: 112

WP_ _. 039695303 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus gallolyticus ] SEQ ID NO: 113

WP_ _. 014334983 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus infantarius ] SEQ ID NO: 114

WP_003099269.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus iniae] SEQ ID NO AHY15608.1 CRISPR-associated protein Csnl [Streptococcus iniae] SEQ ID NO: 116

AHY17476.1 CRISPR-associated protein Csnl [Streptococcus iniae] SEQ ID NO: 117

ESR09100.1 hypothetical protein IUSA1_08595 [Streptococcus iniae IUSA1] SEQ ID NO: 118

AGM98575.1 CRISPR-associated protein Cas9/Csnl, subtype II/NMEMI [Streptococcus iniae SF1]

ALF27331.1 CRISPR-associated protein Csnl [Strept DCOCCUS intermedius ] SEQ ID NO: 120

WP_ _. 018372492 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus massiliensis ] SEQ ID NO

WP_ _. 045618028 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mitis ] SEQ ID NO: 122

WP_ _. 045635197 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mitis ] SEQ ID NO: 123

WP_ _. 002263549 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 124

WP_ _. 002263887 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 125

WP_ _. 002264920 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 126

WP_ _. 002269043 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 127

WP_ _. 002269448 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 128

WP_ _. 002271977 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 129

WP_ _. 002272766 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 130

WP_ _. 002273241 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 131

WP_ _. 002275430 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 132

WP_ _. 002276448 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 133

WP_ _. 002277050 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 134

WP_ _. 002277364 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 135

WP_ _. 002279025 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 136

WP_ _. 002279859 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 137

WP_ _. 002280230 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 138

WP_ _. 002281696 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 139

WP_ _. 002282247 1 type II CRISPR RNA- guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 140

WP_ _. 002282906..1 type II CRISPR RNA--guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 141

WP_ _. 002283846. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 142

WP_ _. 002287255. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 143

WP_ _. 002288990. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 144

WP_ _. 002289641. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 145

WP_ _. 002290427. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 146

WP_ _. 002295753. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 147

WP_ _. 002296423. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 148

WP_ _. 002304487. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 149

WP_ _. 002305844. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 150

WP_ _. 002307203. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 151

WP_ _. 002310390. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 152

WP_ _. 002352408. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 153

WP_ _. 012997688. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 154

WP_ _. 014677909. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 155

WP_ _. 019312892. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 156

WP_ _. 019313659. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 157

WP_ _. 019314093. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 158

WP_ _. 019315370. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 159

WP_ _. 019803776. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 160

WP_ _. 019805234. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 161

WP_ _. 024783594. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 162

WP_ _. 024784288. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 163

WP_ _. 024784666. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 164

WP_ _. 024784894. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 165

WP_ _. 024786433. .1 type II CRISPR RNA- -guided endonuclease Cas9 [ Streptococcus mutans ] SEQ ID NO: 166

WP_049473442.1 CRISPR-associated protein Csnl [Streptococcus mutans] SEQ ID NO: 167

WP_049474547.1 CRISPR-associated protein Csnl [Streptococcus mutans] SEQ ID NO: 168

EMC03581.1 hypothetical protein SMU69_09359 [Streptococcus mutans NLML4] SEQ ID NO: 169

WP_ _. 000428612 1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus oralis] SEQ ID NO: 170

WP_ _. 000428613 1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus oralis] SEQ ID NO: 171

WP_ _. 049523028 1 CRISPR-associated protein Csnl [Streptococcus parasanguinis ] SEQ ID NO 172

WP_ _. 003107102 1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus parauberis] SEQ ID NO: 173

WP_ _. 054279288 1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus phocae] SEQ ID NO: 174

WP_ _. 049531101 1 CRISPR-associated protein Csnl [Streptococcus pseudopneumoniae ] SEQ ID NO: 175

WP_ _. 049538452 1 CRISPR-associated protein Csnl [Streptococcus pseudopneumoniae] SEQ ID NO: 176

WP_ _. 049549711 1 CRISPR-associated protein Csnl [Streptococcus pseudopneumoniae] SEQ ID NO: 177

WP_ _. 007896501 1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus pseudoporcinus ] SEQ ID NO:

EFR44625.1 CRISPR-associated protein, Csnl family [Streptococcus pseudoporcinus SPIN 20026] SEQ ID NO: WP_002897477.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus sanguinis] SEQ ID NO: 180 WP_002906454.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus sanguinis] SEQ ID NO: 181 WP_009729476.1 type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus sp . F0441] SEQ ID NO: 182 CQR24647.1 CRISPR-associated protein [Streptococcus sp . FF10] SEQ ID NO: 183

WP_ _. 000066813 1 type II CRISPR RNA- guided endonuclease Cas9 [Streptococcus sp . M334] SEQ ID NO: 184

WP_ _. 009754323 1 type II CRISPR RNA -guided endonuclease Cas9 [Streptococcus sp . taxon 056] SEQ ID NO : If

WP_ _. 044674937 1 type II CRISPR RNA- guided endonuclease Cas9 [Streptococcus suis ] SEQ ID NO: 186

WP_ _. 044676715 1 type II CRISPR RNA- guided endonuclease Cas9 [Streptococcus suis ] SEQ ID NO: 187

WP_ _. 044680361 1 type II CRISPR RNA- guided endonuclease Cas9 [Streptococcus suis ] SEQ ID NO: 188

WP_ _. 044681799 1 type II CRISPR RNA- guided endonuclease Cas9 [Streptococcus suis ] SEQ ID NO: 189

WP_ _. 049533112 1 CRISPR-associated protein Csnl [Streptococcus suis] SEQ ID NO: 190

WP_ _. 029090905 1 type II CRISPR RNA- guided endonuclease Cas9 [Brochothrix thermosphacta ] SEQ ID NO: 191

WP_ _. 006506696 1 type II CRISPR RNA- guided endonuclease Cas9 [ Catenibacterium mitsuokai] SEQ ID NO: 192

AIT42264.1 Cas9hc: NLS : HA [Cloning vector pYB196] SEQ ID NO: 193

WP_ 034440723 1 type II CRISPR endonuclease Cas9 [ Clostridiales bacterium S5-A11] SEQ ID NO : 194

AKQ21048.1 Cas9 [CRISPR-mediated gene targeting vector p (bhsp68-Cas9) ] SEQ ID NO: 195

WP_ 004636532 1 type II CRISPR RNA- guided endonuclease Cas9 [ Dolosigranulum pigrum] SEQ ID NO : 196

WP_ 002364836 1 MULTISPECIES : type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus ] SEQ ID NO

WP_ 016631044 1 MULTISPECIES : type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus ] SEQ ID NO

EMS75795.1 hypothetical protein H318_ _. 06676 [Enterococcus durans IPLA 655] SEQ ID NO: 199

WP_ 002373311 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 200

WP_ 002378009 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 201

WP_ 002407324 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 202

WP_ 002413717 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 203

WP_ 010775580 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 204

WP_ 010818269 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 205

WP_ 010824395 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 206

WP_ 016622645 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 207

WP_ 033624816 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 208

WP_ 033625576 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 209

WP_ 033789179 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecalis ] SEQ ID NO : 210

WP_ 002310644 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 211

WP_ 002312694 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 212

WP_ 002314015 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 213

WP_ 002320716 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 214

WP_ 002330729 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 215

WP_ 002335161 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 216

WP_ 002345439 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 217

WP_ 034867970 1 type II CRISPR RNA- guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 218

WP_047937432.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus faecium] SEQ ID NO: 219

WP_010720994.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus hirae] SEQ ID NO: 220

WP_010737004.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus hirae] SEQ ID NO: 221

WP_034700478.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus hirae] SEQ ID NO: 222

WP_007209003.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus italicus] SEQ ID NO: 223

WP_023519017.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus mundtii] SEQ ID NO: 224

WP_010770040.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus phoeniculicola ] SEQ ID NO: 225 WP_048604708.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus sp . AMI] SEQ ID NO: 226

WP_010750235.1 type II CRISPR RNA-guided endonuclease Cas9 [Enterococcus villorum] SEQ ID NO: 227

AII16583.1 Cas9 endonuclease [Expression vector pCas9] SEQ ID NO: 228

WP_029073316.1 type II CRISPR RNA-guided endonuclease Cas9 [Kandleria vitulina] SEQ ID NO: 229

WP_031589969.1 type II CRISPR RNA-guided endonuclease Cas9 [Kandleria vitulina] SEQ ID NO: 230

KDA45870.1 CRISPR-associated protein Cas9/Csnl, subtype II/NMEMI [Lactobacillus animalis] SEQ ID NO: 231

•Ji

WP_039099354.1 type II CRISPR RNA-guided endonuclease Cas9 [Lactobacillus curvatus] SEQ ID NO: 232

AKP02966.1 hypothetical protein ABB45_04605 [Lactobacillus farciminis] SEQ ID NO: 233

WP_010991369.1 type II CRISPR RNA-guided endonuclease Cas9 [Listeria innocua] SEQ ID NO: 234

WP_033838504.1 type II CRISPR RNA-guided endonuclease Cas9 [Listeria innocua] SEQ ID NO: 235

EHN60060.1 CRISPR-associated protein, Csnl family [Listeria innocua ATCC 33091] SEQ ID NO: 236

EFR89594.1 crispr-associated protein, Csnl family [Listeria innocua FSL S4-378] SEQ ID NO: 237

WP_038409211.1 type II CRISPR RNA-guided endonuclease Cas9 [Listeria ivanovii] SEQ ID NO: 238

EFR95520.1 crispr-associated protein Csnl [Listeria ivanovii FSL F6-596] SEQ ID NO: 239

WP_ _. 003723650 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 240

WP_ _. 003727705 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 241

WP_ _. 003730785 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 242

WP_ _. 003733029 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 243

WP_ _. 003739838 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 244

WP_ _. 014601172 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 245

WP_ _. 023548323 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 246

WP_ _. 031665337 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 247

WP_ _. 031669209 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 248

WP_ _. 033920898 1 type II CRISPR RNA- guided endonuclease Cas9 [Listeria monocytogenes ] SEQ ID NO: 249

AKI42028.1 CRISPR-associated protein [Listeria monocytogenes] SEQ ID NO: 250

AKI50529.1 CRISPR-associated protein [Listeria monocytogenes] SEQ ID NO: 251

EFR83390.1 crispr-associated protein Csnl [Listeria monocytogenes FSL F2-208] SEQ ID NO: 252

WP_046323366.1 type II CRISPR RNA-guided endonuclease Cas9 [Listeria seeligeri] SEQ ID NO: 253 AKE81011.1 Cas9 [Plant multiplex genome editing vector pYLCRISPR/Cas9Pubi-H] SEQ ID NO: 254

CU082355.1 Uncharacterized protein conserved in bacteria [Roseburia hominis] SEQ ID NO: 255

WP_033162887.1 type II CRISPR RNA-guided endonuclease Cas9 [Sharpea azabuensis] SEQ ID NO: 256 AGZ01981.1 Cas9 endonuclease [synthetic construct] SEQ ID NO: 257

AKA60242.1 nuclease deficient Cas9 [synthetic construct] SEQ ID NO: 258

AKS40380.1 Cas9 [Synthetic plasmid pFC330] SEQ ID NO: 259

4UN5_B Cas9, Chain B, Crystal Structure SEQ ID NO: 260

[0312] Non-limiting examples of suitable deaminase domains are provided.

Human AID

MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELL FLRYISD WDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLR RLHRAGV OIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSROLRRILLPLYEVDDLRDAFR TLGL (SEQ ID NO: 303) (underline: nuclear localization signal; double underline: nuclear export signal)

Mouse AID

MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELL FLRYISD WDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLR RLHRAGV OIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTROLRRILLPLYEVDDLRDAFR MLGF (SEQ ID NO: 271) (underline: nuclear localization signal; double underline: nuclear export signal)

Dog AID

MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELL FLRYISD WDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLR RLHRAGV OIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSROLRRILLPLYEVDDLRDAFR TLGL (SEQ ID NO: 272) (underline: nuclear localization signal; double underline: nuclear export signal)

Bovine AID

MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELL FLRYISD WDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGL RRLHRAG VOIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSROLRRILLPLYEVDDLRDAF RTLGL

(SEQ ID NO: 273) (underline: nuclear localization signal; double underline: nuclear export signal)

Rat AID

MAVGSKPKAALVGPHWERERIWCFLCSTGLGTQQTGQTSRWLRPAATQDPVSPPRSLLMK QRKFLYH FKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFLRYISDWDLDPGR CYRVTWF TSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYFYCWNTFVE NHERTFKA WEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 1072)

Mouse APOBEC-3

MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLH HGVFKNKD NIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQIYRFLATHHNLSLOIFSSRLYNYQOPETQ QNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPC YIPVPSS SSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQL EQFNGQAPL KGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNC AWQLAAFKRORPOLILHIYTSRLYFHWK RPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIK ESWGLQDL VNDFGNLQLGPPMS (SEQ ID NO: 274) (italic: nucleic acid editing domain)

Rat APOBEC-3

MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLH HGVFKNKDN IHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQYLRFLATmiNLSLOlFSS RLYNlRDPENQQ NLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCY IPVPSSSS STLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQ FNGQAPLKG CLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNC AWQLAAFKRORPOLILHIYTSRLYFHWKRPF QKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESW GLQDLVND FGNLQLGPPMS (SEQ ID NO: 275) (italic: nucleic acid editing domain)

Rhesus macaque APOBEC-3G

MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFOGKVYSKAKY//P £MRFL^Wy// KWRQLHHDQEYKVTWYVSWSPCTRC ANSYATFLAKOPKYTLTIFYARLYYFWKPOYQQALRILCQKRG GPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFT SNFNNKP WVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGR/MSLCFLDLWMTLD Geeray rCFreWSPCraCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMN YSEFEYCW DTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (SEQ ID NO: 276) (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

Chimpanzee APOBEC-3G

MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS KLKYZ/P£M RFFHWFSKWRKLHRDQEYEWWYISWSPCTKCTROY ATFLAEDPKYTLTIFYARLYYFWOPOYQEALRS LCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSM DPPTFTSN FNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGR/MSLCFLDy WWTiLD Li/eDyRyrCFreWSPCraCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKA GAKISIMTYSE FKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO: 277) (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

Green monkey APOBEC-3 G

MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYP EAKDZ/P£M KFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSY ATFLAEDPKYTLTIFYARLYYFWKPOYQQALRI LCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVM DPGTFTS NFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGR/MSLCFLD LWMrL DDeeyRyrCFreWSPCraCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDG AKIAVMNYS EFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (SEQ ID NO:278) (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) Human APOBEC-3G

MKPHFRNTVERMYRPTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLPAKIFRGQVYS ELKYZ/P£M RFFHWFSKWRKLHRDQEYEWWYISWSPCTKCTROMATFLAEOPKVTLTIFV ARLYYFWOPOYQEALRS LCQKRPGPRATMKIMNYPEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSM PPPTFTFN FNNEPWVRGRHETYLCYEVERMHNPTWVLLNQRRGFLCNQAPHKHGFLEGR/MSLCFLDW PiTWTLD LDeDyRyrCFreWSPCraCAQEMAKFISKNKHVSLCIFTARIYPPQGRCQEGLRTLAEAG AKISIMTYSE FKHCWPTFVPHQGCPFQPWPGLPEHSQPLSGRLRAILQNQEN (SEQ IP NO: 279) (italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

Human APOBEC-3F

MKPHFRNTVERMYRPTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLPAKIFRGQVYS QPEH/MSM CFLSWFCGNQLPAYKCFQITWFVSWTPCPDCV AKLAEFLAEHPNVTLTISAARLYYYWERPYRRALCRL SQAGARVKIMPPEEFAYCWENFVYSEGQPFMPWYKFPPNYAFLHRTLKEILRNPMEAMYP HIFYFHF KNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVPPETHCiM^CFL^WCDD/L^P^ TTVreVT WmWSPCP£CAGEVAEFLARHSNVNLTIFTARLYYFWPTPYQEGLRSLSQEGASVEIMGY KPFKYCW ENFVYNPPEPFKPWKGLKYNFLFLPSKLQEILE (SEQ IP NO: 280) (italic: nucleic acid editing domain)

Human APOBEC-3B

MNPQIRNPMERMYRPTFYPNFENEPILYGRSYTWLCYEVKIKRGRSNLLWPTGVFRGQVY FKPQY/M EMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCV AKLAEFLSEHPNVTLTISAARLYYYWERDYRRALC RLSQAGARVTIMPYEEFAYCWENFVYNEGQQFMPWYKFPENYAFLHRTLKEILRYLMPPP TFTFNFN NPPLVLRRRQTYLCYEVERLPNGTWVLMPQHMGFLCNEAKNLLCGFYG^/MSL^FLDLyP ^LeLDPA e/yRy7W/5WSPCraWGCAGEVRAFLQENTHVRLRIFAARIYPYPPLYKEALQMLRPAGA QVSIMTYP EFEYCWPTFVYRQGCPFQPWPGLEEHSQALSGRLRAILQNQGN (SEQ IP NO: 281) (italic: nucleic acid editing domain)

Rat APOBEC-3B:

MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCY EVNGMP

CALPVPLRQGVFRKQGHIHAELCFIYWFHPKVLRVLSPMEEFKVTWYMSWSPCSKCA EQVARFLAAH

RNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMPLPEFKKCWNKFVPNPGQP FRPWMRLRINF

SFYPCKLQEIFSRMNLLREPVFYLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQE PLKGYLLYKK

GEQHVEILFLEKMRSMELSQVRITCYLTWSPCPNCARQLAAFKKPHPPLILRIYTSR LYFYWRKKFQKG

LCTLWRSGIHVPVMPLPQFAPCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESW GL (SEQ ID

NO: 1073)

Bovine APOBEC-3B:

PGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFG NQPRV PAPYYRRKTYLCYQLKQRNPLTLPRGCFRNKKQRHAEIRFIPKINSLPLNPSQSYKIICY ITWSPCPNCA NELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFV DNQSRPFQP WDKLEQYSASIRRRLQRILTAPI (SEQ ID NO: 1074)

Chimpanzee APOBEC-3B:

MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMY SQPEHH

AEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARL YYYWERDYRR

ALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIR HLMDPDTFTF

NFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELR FLDLVPSLQ

LDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEAL QMLRDAGAQVS

IMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRP PPPPQSPGPCLP

LCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSL TSCSIQPPCSSRIRETEG

WASVSKEGRDLG (SEQ ID NO: 1075) Human APOBEC-3C:

MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVD SETHO/ AERCFLSWFCDDILSPNTKYQWWYTSWSPCPDCAGEV AEFLARHSNVNLTIFTARLYYFQYPCYQEGLR SLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ (SEQ ID NO: 282) (italic: nucleic acid editing domain)

Gorilla APOBEC-3C:

MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVD SETHCH AERCFLSWFCDDILSPNTNYQVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYF QDTDYQEG LRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFKPWKGLKYNFRFLKRRLQEILE (SEQ ID NO: 1076)

Human APOBEC-3A:

MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAK NLLCGFY GRHAELRFLDLVPSLQLDPAQIYRWWFISWSPCFSWGCAGEVRAFLQEmHVRLRlFAARl YOYOPLYKE ALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQG N (SEQ ID NO: 283) (italic: nucleic acid editing domain)

Rhesus macaque APOBEC-3A:

MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGFLCN KAKNVP CGDYGCHVELRFLCEVPSWQLDPAQTYRVTWFISWSPCFRRGCAGQVRVFLQENKHVRLR IFAARIYD YDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHSQALSGRLR AILQNQG

N (SEQ ID NO: 1077) Bovine APOBEC-3A:

MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPCHAELY FLGKIHS WNLDRNQHYRLTCFISWSPCYDCAQKLTTFLKENHHISLHILASRIYTHNRFGCHQSGLC ELQAAGARI TIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKSQALCTELQAILKTQQN (SEQ ID NO: 1078)

Human APOBEC-3H:

MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKC/MS/CFW S/^MGLD SrgCyevrCyLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGS QVPVEVMG FPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCD AEV (SEQ ID NO: 284) (italic: nucleic acid editing domain)

Rhesus macaque APOBEC-3H:

MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFI NKIKSMGL DETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCG SQVPVEVM GLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQ LGPVTPSSSIR NSR (SEQ ID NO: 1079)

Human APOBEC-3D

MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVL PKRQSNH

RQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHP NVTLTISAARLY

YYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASL HRTLKEIL

RNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPE THC/M^CFL

SWFCDD/LSPNrNy^VTWrSWSPCPSCAGEVAEFLARHSNVNLTIFTARLCYFWDTD YQEGLCSLSQEG

ASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (SEQ ID NO: 285) (italic: nucleic acid editing domain)

Human APOBEC-1

MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTN HVEVNFIKK FTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQ GLRDLVNS GVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISR RWQNHLTF FRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 286)

Mouse APOBEC-1

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRK£7TLLYEINWGGRHSVWRHTSQNTS NHVEVNFLEK FTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQ GLRDLISSGVTI QIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQP QLTFFTITL QTCHYQRIPPHLLWATGLK (SEQ ID NO: 287) Rat APOBEC-1

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRK£7TLLYEINWGGRHSIWRHTSQNTN KHVEVNFIEKF TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQG LRDLISSGVTI QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQP QLTFFTIALQ SCHYQRLPPHILWATGLK (SEQ ID NO: 288)

Human APOBEC-2:

MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVE YSSGRNKTF LCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCA ACADRIIK TLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEG ESKAFQPW EDIQENFLYYEEKLADILK (SEQ ID NO: 1080)

Mouse APOBEC-2:

MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVE YSSGRNKTF LCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCA ACADRIL KTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEE GESKAFEP WEDIQENFLYYEEKLADILK (SEQ ID NO: 1081)

Rat APOBEC-2:

MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVE YSSGRNKTF LCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCA ACADRIL KTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEE GESKAFEP WEDIQENFLYYEEKLADILK (SEQ ID NO: 1082)

Bovine APOBEC-2:

MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVE YSSGRNKTF LCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCA ACADRIV KTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEE GESKAFEP WEDIQENFLYYEEKLADILK (SEQ ID NO: 1083)

Petromyzon marinus CDA1 (pmCDAl)

MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSG TERGIHAE IFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYY EKNARNQI GLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQ VKILHTTK SPAV (SEQ ID NO: 289)

Human APOBEC3G D316R_D317R MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS ELKYHPEM

RFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYY FWDPDYQEAL

RSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEI LRHSMDPPTFTF

NFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELC FLDVIPFWK

LDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRT LAEAGAKISIMT

YSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO: 290)

Human APOBEC3G chain A

MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGR HAELCFL DVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQE GLRTLAEAG AKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ (SEQ ID NO: 291)

Human APOBEC3G chain A D120R_D121R

MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGR HAELCFL DVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQE GLRTLAEAG AKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ (SEQ ID NO: 292)

[0313] Non-limiting examples of fusion proteins/nucleobase editors are provided.

His ₆ -rAPOBECl-XTEN-dCas9 for Escherichia coli expression (SEQ ID NO: 293)

MGSSHHHHHHMSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGR HSIWRHTSONTN

KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYI ARLYHHADPRNRQG

LRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIIL GLPPCLNILRRKO

PQLTFFTIALOSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGT NSVGWAVITDEYK

VPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQ EIFSNEMAKVDD

SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR LIYLALAHMIKFR

GHFLIEGDLNPDNSDVDKLFIQLVOTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKN

GLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDOYADLFLA AKNLSDAILLSD

ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVROQLPEKYKEIFFDQSKNGYAG YIDGGASOEEFY

KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFY PFLKDNREKIEKIL

TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSL

LYEYFTVYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEIS

GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED IOKAQVSGOGDS

LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGOKN SRERMKRIEEGI

KELGSOILKEHPVENTQLONEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQS FLKDDSIDNKV

LTRSDKNRGKSDNVPSEEVVKKMKNYWROLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVE

TRQITKHVAOILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN YHHAHDAYLNA

VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKR

PLIETNGETGEIVWDKGRDFATVRKVLSMPOVNIVKKTEVQTGGFSKESILPKRNSD KLIARKKDWDPK

KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG YKEVKKDLIIKLP

KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEOKO LFVEQHKHYL

DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYT

STKEVLDATLIHQSITGLYETRIDLSOLGGDSGGSPKKKRKV [0314] rAPOBECl-XTEN-dCas9-NLS for Mammalian expression (SEQ ID NO: 294)

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQN TNKHVEVNFIEKF

TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRN RQGLRDLISSGVTI

QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRR KQPQLTFFTIALQ

SCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDE YKVPSKKFKVLG

NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFL

VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLN

PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALS

LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITK

APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEE FYKFIKPILEKM

DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGP

LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYN

ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE ISGVEDRFNASL

GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM KQLKRRRYTGWG

RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGS

PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG IKELGSQILKEH

PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV LTRSDKNRGKS

DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV VGTALIKKYPK

LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR PLIETNGETGEIV

WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVA

YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGR

KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKR

VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRY TSTKEVLDATLIH

QSITGLYETRIDLSQLGGDSGGSPKKKRKV

[0315] hAPOBEC l-XTEN-dCas9-NLS for Mammalian expression (SEQ ID NO: 295)

MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEI KWGMSRKIWRSSGKNTTNHVEVN FIKKF

TSERDFHPSMSCSITWFLSWSPCWECSQAI REFLSRH PGVTLVIYVARLFWHMDQQNRQGLRDLVNSG

VTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCI ILSLPPCLKISRRWQNHLTFFR

LHLQNCHYQTIPPHILLATGLI HPSVAWRSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVP

SKKFKVLGNTDRHSI KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGN IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL

N PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSL

GLTPN FKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAI LLSDI LRVNTEITKAPLS

ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYI DGGASQEEFYKFIKPI LEKMDGTEELL

VKLN REDLLRKQRTFDNGSIPHQIHLGELHAI LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA

WMTRKSEETITPWN FEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYN ELTKVKYVTEG

MRKPAFLSGEQKKAIVDLLFKTN RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDF

LDN EEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLI NGIRDKQSGK

TI LDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAI KKGI LQTVKVVDELV

KVMGRHKPEN IVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN EKLYLYYLQN

GRDMYVDQELDIN RLSDYDVDAIVPQSFLKDDSI DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL

LNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL

KSKLVSDFRKDFQFYKVREIN NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI

GKATAKYFFYSN IMN FFKTEITLANGEI RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEV

QTGGFSKESI LPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RS

SFEKN PIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL

KGSPEDNEQKQLFVEQHKHYLDEII EQISEFSKRVI LADAN LDKVLSAYN KHRDKPIREQAEN II HLFTLTNL

GAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKKRKV [0316] rAPOBECl-XTEN-dCas9-UGI-NLS (SEQ ID NO: 296)

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK HVEVNFIEKF

TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRN RQGLRDLISSGVTI

QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRR KQPQLTFFTIALQ

SCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDE YKVPSKKFKVLG

NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFL

VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLN

PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALS

LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITK

APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEE FYKFIKPILEKM

DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGP

LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYN

ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE ISGVEDRFNASL

GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM KQLKRRRYTGWG

RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGS

PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG IKELGSQILKEH

PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV LTRSDKNRGKS

DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV VGTALIKKYPK

LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR PLIETNGETGEIV

WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVA

YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGR

KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKR

VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRY TSTKEVLDATLIH

QSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPESDILVHTAYDES

TDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV

[0317] rAPOBECl-XTEN-Cas9 nickase-UGI-NLS (BE3, SEQ ID NO: 297)

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK HVEVNFIEKF

TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRN RQGLRDLISSGVTI

QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRR KQPQLTFFTIALQ

SCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDE YKVPSKKFKVLG

NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFL

VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLN

PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALS

LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITK

APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEE FYKFIKPILEKM

DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGP

LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYN

ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE ISGVEDRFNASL

GTYHDLLKIIKDKDFLDNEENEDILEDIVLT1TLFEDREMIEERLKTYAHLFDDKVM KQLKRRRYTGWG

RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGS

PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG IKELGSQILKEH

PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKS

DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV VGTALIKKYPK

LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR PLIETNGETGEIV

WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVA

YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGR

KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKR

VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRY TSTKEVLDATLIH

QSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPESDILVHTAYDES

TDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV

[0318] pmCDAl-XTEN-dCas9-UGI (bacteria) (SEQ ID NO: 298) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNK

PQS GTERGIH AEIFS IRKVEE YLRDNPGQFTINW YS S WSPC ADC AEKILEW YNQELRG

NGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQ

LNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAVSGSETPGTSESATPESDKK YS

IGLAIGTNS VGW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET AE ATRL

KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN I

VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS

DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG L

FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK

NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FD

QS KNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IP

HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK S

EETITPWNFEE V VD KG AS AQS FIERMTNFD KNLPNEKVLPKHS LL YE YFT V YNELTK

VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV

EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HL

FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH

DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK

PENrVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY L

YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN

VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR

QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF

YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV K

KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG

KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG RK

RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD

EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF KYF

DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSMTNLSDIIEKET GK

QLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALV I

QDSNGENKIKML

[0319] pmCDAl-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 299):

MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNK

PQS GTERGIH AEIFS IRKVEE YLRDNPGQFTINW YS S WSPC ADC AEKILEW YNQELRG

NGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQ

LNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAVSGSETPGTSESATPESDKK YS

IGLAIGTNS VGW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET AE ATRL

KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN I

VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS

DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG L

FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK

NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FD

QS KNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IP

HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK S

EETITPWNFEE VVD KG AS AQS FIERMTNFD KNLPNEKVLPKHS LL YE YFT V YNELTK

VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV

EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HL

FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH

DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL

YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN

VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR

QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF

YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV K

KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG

KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG RK

RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD

EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF KYF

DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETG KQL

VIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQ D

S NGENKIKMLS GGS PKKKRKV

[0320] huAPOBEC3G-XTEN-dCas9-UGI (bacteria) (SEQ ID NO: 300)

MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG

FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLC I

FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLD

EHS QDLS GRLR AILQS GS ETPGTS ES ATPES DKKYS IGLAIGTNS VG W A VITDE YKVPS

KKFKVLGNTDRHS IKKNLIGALLFDS GET AE ATRLKRT ARRRYTRRKNRIC YLQEIFS

NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVD

STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI N

AS G VD AKAILS ARLS KS RRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKS NFDLAE

DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL S

ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF

IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF LK

DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF I

ERMTNFDKNLPNEKVLPKHS LLYE YFTV YNELTKVKYVTEGMRKPAFLS GEQKKAI

VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD F

LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS

RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL H

EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR

ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD

YDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK

LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND

KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL

ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP L

IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL I

ARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEK

NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV

NFLYLAS H YEKLKGS PEDNEQKQLF VEQHKH YLDEIIEQIS EFS KRVILAD ANLD KVL

SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQ

SITGLYETRIDLSQLGGDSGGSMTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPE

S DILVHT A YDES TDEN VMLLTS D APE YKPW ALVIQDS NGENKIKML

[0321] huAPOBEC3G-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 301)

MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLD

EHS QDLS GRLR AILQS GS ETPGTS ES ATPES DKKYS IGLAIGTNS VG W A VITDE YKVPS

KKFKVLGNTDRHS IKKNLIGALLFDS GET AE ATRLKRT ARRRYTRRKNRIC YLQEIFS

NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVD

STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI N

AS G VD AKAILS ARLS KS RRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKS NFDLAE

DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL S

ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF

IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF LK

DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF I

ERMTNFDKNLPNEKVLPKHS LLYE YFTV YNELTKVKYVTEGMRKPAFLS GEQKKAI

VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD F

LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS

RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL H

EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR

ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD

YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK

LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND

KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL

ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP L

IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL I

ARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEK

NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV

NFLYLAS H YEKLKGS PEDNEQKQLF VEQHKH YLDEIIEQIS EFS KRVILAD ANLD KVL

SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQ

SITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PES

DILVHT A YDES TDEN VMLLTS D APE YKPW ALVIQDS NGENKIKMLS GGS PKKKRKV

[0322] huAPOBEC3G (D316R_D317R)-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 302)

MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG

FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLC I

FTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDE

HS QDLS GRLRAILQS GS ETPGTS ES ATPES DKKYS IGLAIGTNS VGW A VITDE YKVPS K

KFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS N

EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS

TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN A

S GVD AKAILS ARLS KS RRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKS NFDLAED

AKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS A

S MIKR YDEHHQDLTLLKALVRQQLPEKYKEIFFDQS KNG Y AG YID GG AS QEEF YKFI

KPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL K

DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF I

ERMTNFDKNLPNEKVLPKHS LLYE YFTV YNELTKVKYVTEGMRKPAFLS GEQKKAI

VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD F

LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS

RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL H

EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR

ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK

LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND

KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL

ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP L

IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL I

ARKKD WDPKKYGGFDS PT V A YS VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEK

NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV

NFLYLAS H YEKLKGS PEDNEQKQLF VEQHKH YLDEIIEQIS EFS KRVILAD ANLD KVL

SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQ

SITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PES

DILVHT A YDES TDEN VMLLTS D APE YKPW ALVIQDS NGENKIKMLS GGS PKKKRKV

Base Editor 4 (BE4; APOBECl-linker(32 aa)-Cas9n(D10A)-linker(9 aa)-UGI-linker(9 aa)-

UGI)

MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK HVEVNFIEKF

TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRN RQGLRDLISSGVTI

QIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRR KQPQLTFFTIALQ

SCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIG LAIGTNSVGWAVI

TDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR ICYLQEIFSNEM

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALA

HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARL SKSRRLENLIAQL

PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLS

DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGA

SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILR RQEDFYPFLKDNR

EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNFDKNLPNEK

VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQ LKEDYFKKIEC

FDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM IEERLKTYAHLFDD

KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQV

SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERM

KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDV DHIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGG LSELDKAGFI

KRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAH

DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI MNFFKTEITLA

NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESI LPKRNSDKLIARK

KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVK

KDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVE

QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTI

DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKE TGKQLVIQESILML

PEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIK MLSGGSGGSGGS

TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLL TSDAPEYKPWALVI

QDSNGENKIKMLSGGSPKKKRK (SEQ ID NO: 1084)

Example 2: Anti-Cancer Vaccination Using CRISPR-Cas9 Genome/base-editing

Technologies

[0323] Described herein are new methods to stimulate the immune system to treat tumors and prevent metastatic lesions. By turning the genome and proteome of the malignant cells into a personalized endogenous anti-cancer vaccine in vivo.

[0324] Provided herein is a new immuno-oncology methodology to raise robust T-cell and B-cell mediated immune responses against tumor- specific proteins, which are otherwise tolerated as "self by the immune system of a cancer patient 1- ^" 3 (Figure 1, Tables 1-3). This methodology is uniquely suited for programmable CRISPR-Cas9 genome- and base-editing tools, ^4"10 exploited to alter the translated sequences of tumor specific genes ¹¹ to produce highly immunogenic heteroclitic and cryptic peptide epitopes in situ (Tables 5 and 7, Figure 1). Heteroclitic epitopes are altered versions of endogenous peptide sequences engineered to elicit potent immune reactions through the MHC-I and MHC-II antigen presentation pathways, 12 which also produce cross -reactive responses towards the parent wild-type peptide sequences (Figure 2A) 2 ' 13 ' 14. For example, the peptide epitope EAAGIGILTV from the melanocyte differentiation and melanoma marker MART-1 ^26"36 is weakly immunogenic, whereas vaccination with a similar peptide engineered with a hydrophobic residue ¹⁵ ' ¹⁶ on the MHC-anchor position MART-1 (27L) ELAGIGILTV promotes robust T-cell immune responses against melanoma (Table 5). 17 ' 18 Cryptic epitopes arise from non-translated genomic sequences through processes that are elevated in cancer cells, such as aberrant mRNA splicing, alternative open-reading frames (ORFs), and deglycosidation of proteins. ¹⁹

For example, LAGE-1 immunogenic antigens are expressed from ORF-2 20 ' 21 of the gene NY-ESO- 122-24 (Table 7). Introduction of these immunogenic protein sequences using genome/base editing is designed to break "self-tolerance to cancer-specific antigens, 1- ^" 3 which is known drive the infiltration of immune cells into the tumor promoting the recognition of malignant cells as foreign through multiple mechanisms (Figure 3). ²⁵ ' ²⁶ Anticancer vaccination using genome/base-editing is rendered cancer- specific by targeting genes that are preferentially or exclusively expressed by tumor cells to prevent autoimmunity side effects. 2 ' 27 Anti-cancer vaccination strategies could be particularly useful for the treatment of melanoma (Table 1), 28- ^" 30 as well as colorectal tumors, stomach cancer, and other highly mutagenized cancers that accumulate non-synonymous hitchhiker mutations with high frequencies (Figure 4). 31 In such cases, spreading of the adaptive immune response towards translated "neo-epitopes" 32 ' 33 that are unique to the cell lineage facilitate remission34 and prevent metastatic lesions (abscopal effect) 35- ^" 39 (Figure 3). Importantly, the recently FDA- approved checkpoint inhibitors (anti-PDl and anti-CTLA4 antibodies), which lower the

27 40

threshold for T-cell stimulation, ' have shown promise for co-administration with anticancer vaccines, ^41"46 and could enhance the clinical effectiveness of immunization against a broader assortment of cancer types. 47 ' 48

[0325] The heteroclitic and cryptic epitopes programmed by genome/base-editing may be personalized to match each patient's malignancy and immune system, or alternatively a guide-RNA cocktail can be developed to engage the most frequent HLA allele supertypes that broadly cover the human population (Figure 2B). Recent advances for the delivery of genome editing tools are enabling for anti-cancer vaccination. These methods include intracellular delivery using electroporation, ⁴⁹ ' ⁵⁰ viral vectors, ⁵¹ ' ⁵² cell penetrating peptides, ^" liposomes, ⁵⁵ ' ⁵⁶ polymers, ⁵⁷ membrane deformation, ⁵⁸ and nanoparticles ⁵⁹ ; and the types of cargo include RNA transcripts, DNA expression vectors, or Cas9 protein-guide RNA complexes purified, ⁶⁰ or within cationic lipid vesicles. ⁶¹ ' ⁶² Therefore, the vaccination treatments could be potentially performed in vivo directly on tumor cells, ⁶³ the tissues that originated the tumor, ⁶⁴ or alternatively ex vivo for re-injection of irradiated whole-cell vaccines. 65-67

[0326] Also provided herein are numerous specific examples of genomic target sites in tumor-associated genes (Tables 1-3), and the guide-RNAs designed to program the alteration of these translated sequences (Tables 5 and 7), in order to replicate or closely mimic known epitopes that have literature or pre-clinical precedent. The genome editing reactions were designed for one of the CRISPR/Cas9 tools: (i) "base editors" that catalyze chemical reactions on nucleobases (e.g. cytidine deaminase-Cas9 fusion4); or (ii) engineered nucleases with DNA cutting activity (e.g. WT Cas9, 5- ^" 7 Cas9 nickases 8 or Fokl-nuclease-dCas9 fusions ⁹ ' ¹⁰ ). Examples of other potentially useful genome-editing reactions to alter cancer- specific genes to produce heteroclitic/cryptic epitopes are shown in Tables 5 and 7. By extension, Cas9 tools and Homology-Directed Repair (HDR) pathways may also be exploited to introduce heteroclitic epitopes through DNA templates by lowering the rate of indels using several techniques. ^68"70 Finally, to expand the repertoire of heteroclitic and cryptic epitopes in an unbiased high-throughput manner, the aforementioned tools could be used to screen libraries of guide-RNAs targeting all PAM sites across a tumor-associated genes of interest, which can be replicated using the genome/base-editing reactions shown in Tables 5 and 7. 71 ' 72

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EQUIVALENTS AND SCOPE

[0327] In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0328] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0329] It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0330] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0331] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.