Base editing enzymes

Abstract

The present disclosure provides for engineered base editing system comprising endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof, wherein the engineered base editing system comprises a base editor and an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence.

Description

Attorney Docket No : 00010.037.1801BASE EDITING ENZYMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 704.460, filed on October 7, 2024, the entire content of which is hereby incorporated by reference herein in its entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 30, 2025. is named 00010_037_1801_SL.xml and is 1,313,784 bytes in size.BACKGROUND

[0003] Base editors (BEs) are gene editors that directly modify a nucleobase to install point mutations into cellular nucleic acid molecules without generating double-stranded DNA (dsDNA) breaks. Adenine base editors (ABEs) comprise a nickase (nuclease with one of two catalytic sites disabled) fused to an adenosine deaminase (ADA) enzyme. Improved base editors are needed that allow for facile AAV packaging for use as therapeutics.SUMMARY

[0004] Described herein, in certain embodiments, are engineered base editing systems, comprising: a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%. 98%. 99% or 100% identity to any one of SEQ ID NOs: 210, 356. 182-209. 211-355. 357. and 358; and b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity’ to any one of SEQ ID NOs: 210, 356. 182-209. 211-355. 357, and 358. In some embodiments, the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 210, 356, 182-209, 211-355, 357, and 358. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 31, 177, 2-8, 10-30, 32-176, 178, and 179. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 31, 177, 2-8, 10-30, 32-176, 178, and 179. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100% identity’ to any one of SEQ ID- 1 -#137172Attomey Docket No : 00010.037.1801 NOs: 31, 177, 2-8, 10-30, 32-176, 178, and 179. In some embodiments, the base editor comprises a sequence having at least 70%. 75%. 80%. 85%. 90%. 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 210, 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 210, 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 210, 272. 281, 289, 350, and 351.

[0005] In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 31, 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 31, 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 31 , 93, 102, 1 10, 171 , and 172.

[0006] In some embodiments, the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity' to any one of SEQ ID NOs: 212. 213, 216, 229, 242, 255, 268. 276, 277, 284, 285. 293, 331, and 356. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 212, 213, 216, 229, 242, 255, 268, 276, 277, 284, 285, 293, 331, and 356. In some embodiments, the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 212, 213, 216, 229, 242, 255, 268. 276, 277, 284. 285, 293, 331, and 356. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%. 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 33, 34, 37, 50, 63, 76, 89, 97, 98, 105, 106, 114, 152, and 177. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 33, 34, 37, 50, 63, 76, 89, 97, 98. 105, 106, 114. 152, and 177. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 33, 34, 37, 50, 63, 76, 89, 97, 98, 105, 106, 114, 152, and 177.

[0007] In some embodiments, the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%. 99% or 100% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having 100% sequence identity' to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%. 75%, 80%, 85%, 90%, 95%, 98%. 99% or 100% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 2- 8. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100%- 2 -#137172Attomey Docket No : 00010.037.1801 identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 189-21 1. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity7to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity7to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity7to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity' to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity7to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor comprises a deaminase. In some embodiments, the deaminase binds non-covalently to the endonuclease. In some embodiments, the deaminase is covalently linked to the endonuclease. In some embodiments, the deaminase is fused to the endonuclease. In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the endonuclease binds non-covalently to the- 3 -#137172Attomey Docket No : 00010.037.1801 engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 180 or 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity' to SEQ ID NO: 180 or 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to SEQ ID NO: 180 or 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO: 180 or 181. In some embodiments, the base editor comprises a nickase domain. In some embodiments, the nickase comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 362, residue 13 relative to SEQ ID NOs: 363, 364. or 366. residue 12 relative to SEQ ID NO: 365, residue 17 relative to SEQ ID NO: 367, residue 23 relative to SEQ ID NO: 368, or residue 10 relative to SEQ ID NO: 369, or any combination thereof. In some embodiments, the base editor further comprises a uracil DNA glycosylase inhibitor sequence. In some embodiments, the base editor further comprises a FAM72A sequence. In some embodiments, the FAM72A sequence has at least 80% identity’ to SEQ ID NO: 370.

[0008] Described herein, in certain embodiments, are nucleic acids encoding the engineered base editing system described herein.

[0009] Described herein, in certain embodiments, are vectors comprising the nucleic acids described herein. In some embodiments, the vector is a plasmid, a mini circle, a CELiD, an adeno- associated virus (AAV) derived virion, a lentivirus, or an adenovirus.

[0010] Described herein, in certain embodiments, are cells comprising the engineered base editing system described herein, the nucleic acids described herein, or the vectors described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is an A549, HEK-293. HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary7cell, or a derivative thereof. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0011] Described herein, in certain embodiments, are methods for modifying a target nucleic acid sequence, comprising: contacting the target nucleic acid sequence using the engineered base editing system described herein. In some embodiments, modifying the target nucleic acid sequence comprises converting an adenine to a guanine in the target nucleic acid sequence. In some- 4 -#137172Attomey Docket No : 00010.037.1801 embodiments, modifying the target nucleic acid sequence comprises converting a cytosine to a uracil in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence comprises deoxyribonucleic acid (DNA). In some embodiments, the target nucleic acid sequence comprises ribonucleic acid (RNA). In some embodiments, the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, the target nucleic acid sequence is modified in vitro. In some embodiments, the target nucleic acid sequence is modified in vivo. In some embodiments, the target nucleic acid sequence is modified ex vivo. In some embodiments, the target nucleic acid sequence is modified within a cell. In some embodiments, the cell is a prokary otic cell, a bacterial cell, a eukary otic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, a human cell, or a primary cell.

[0012] Described herein, in certain embodiments, are methods of modifying a nucleic acid encoding AAVS 1 comprising contacting the nucleic acid sequence encoding AAVS1 with an engineered base editing system, said base editing system comprising: a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%. 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-358; and b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity' to SEQ ID NO: 180 or SEQ ID NO: 181.

[0013] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are show n and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the disclosure are set forth with particularity' in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also '‘Figure” and “FIG.” herein), of which:

[0015] FIGs. 1A-1C depict a cell fluorescence screen to identify improved ABE variants. FIG. 1A depicts a schematic of HEK293T cell fluorescence screen. FIG. IB depicts the molecular basis of the cell fluorescence screen. ABE converts the target adenine in an in-frame stop codon of the- 5 -#137172Attomey Docket No : 00010.037.1801 target (mApoAl or AAVS1) to a TGG sense codon enabling expression of the mCherry protein. FIG. IB discloses SEQ ID Nos: 443-448. respectively, in order of appearance. FIG. 1C depicts improved ADA MG68-4 variants (SEQ ID NOs. 2-7) identified from the bacterial chloramphenicol selection characterized in a HEK293T cell fluorescence screen. The activity correlation was assessed by plotting the fraction of dually transfected HEK293T cells expressing the edited target mCherry product against the logarithmic number of E. coll cells that survived 320 pg / mL chloramphenicol.

[0016] FIGs. 2A-2B depict a domain organization of the small base editors where FIG. 2A depicts a deaminase MG68-4 fused on either end of the nickase chassis or inlaid within the nickase and FIG. 2B shows a pooled mRNA screening approach providing base editor pools of 2-6 mRNA tested across 16 nuclease validating targets at multiple loci.

[0017] FIG. 3 depicts results from the ABE pooled mRNA screen showing the maximum base editing activity observed with each pool (Tables 3-5 provide details regarding ABE) at one target site.

[0018] FIGs. 4A-4B depict a max A to G conversion by small base editors targeting a nRC PAM. FIG. 4A shows the max percent A to G editing of each ABE pool at five AAVS1 targets screened by the nucleofection in K562 in replicates of two. FIG. 4B show s the max percent A to G editing of individual ABEs after unpooling with highest editing guide from the pooled screen.

[0019] FIGs. 5A-5B depict a max A to G conversion by small base editors targeting a nAR PAM. FIG. 5A shows the max percent A to G editing of two ABE pools at 9 targets in TRAC and 7 targets in TTR screened by nucleofection in K562 in replicates of two. FIG. 5B shows the max percent A to G editing of individual ABEs after unpooling with highest editing guides from the pooled screen.

[0020] FIGs. 6A-6B depict a max A to G conversion by small base editors targeting a nGG PAM. FIG. 6A shows the max percent A to G editing of each ABE pool at 12 targets in AAVS1, 3 Rosa targets, and 1 TRAC target screened by nucleofection in K562 in replicates of tw o. FIG. 6B show's the max percent A to G editing of individual ABEs (ABE-118 (amino acid sequence denoted by SEQ ID NO: 215), ABE-131 (amino acid sequence denoted by SEQ ID NO: 228), ABE-144 (amino acid sequence denoted by SEQ ID NO: 241), ABE- 157 (ammo acid sequence denoted by SEQ ID NO: 254), ABE-119 (amino acid sequence denoted by SEQ ID NO: 216), ABE-115 (amino acid sequence denoted by SEQ ID NO: 212), ABE-132 (amino acid sequence denoted by SEQ ID NO: 229), ABE-145 (amino acid sequence denoted by SEQ ID NO: 242), ABE-158 (amino acid sequence denoted by SEQ ID NO: 255), ABE- 123 (amino acid sequence denoted by SEQ ID NO: 220), and ABE-163 (amino acid sequence denoted by SEQ ID NO: 260)) after unpooling with highest editing guides from the pooled screen.- 6 -#137172Attorney Docket No : 00010.037.1801

[0021] FIGs. 7A-7C depict domain-walking of improved monomeric ADA MG68-4 variants in MG102-71 and MG34-29, and arginine scanning in MG102-71 nickase. FIG. 7A shows the activity7of MG102-71 domain-walking ABE variants at human AAVS1. FIG. 7B shows the activity7of MG102-71 ABE arginine-scanning variants. The threshold for improvement determination (dotted line) is based on ABE-192 (amino acid sequence denoted by SEQ ID NO: 289). FIG. 7C shows the activity of MG34-29 ABE domain-walking variants at human AAVS1. The threshold for improvement determination (dotted line) is based on ABE- 158 (amino acid sequence denoted by' SEQ ID NO: 255).

[0022] FIG. 8 depicts a max A to G conversion by small base editors ABE-158 (amino acid sequence denoted by SEQ ID NO: 255), ABE-119 (amino acid sequence denoted by SEQ ID NO: 216). and ABE- 163 (amino acid sequence denoted by SEQ ID NO: 260) targeting an nGG PAM at the AAVS 1 locus in SkM cells (guide E7) showing the max percent A to G editing was tested by the lipofection in SkM in replicates of two. A representative replicate is show n here.

[0023] FIGs. 9A-9B depict base editing data in SkM cells for rationally engineered small base editor variants for MG34-29 ABE (FIG. 9A) (ABE-267, amino acid sequence denoted by SEQ ID NO: 352; ABE-272, amino acid sequence denoted by SEQ ID NO: 357; ABE-269, amino acid sequence denoted by SEQ ID NO: 354; ABE-218, amino acid sequence denoted by SEQ ID NO: 311; ABE-270, amino acid sequence denoted by SEQ ID NO: 355; ABE-271, amino acid sequence denoted by SEQ ID NO; 356; ABE-211, amino acid sequence denoted by SEQ ID NO; 304; ABE- 268, amino acid sequence denoted by SEQ ID NO: 353; ABE-273, amino acid sequence denoted by SEQ ID NO: 358) and MG102-71 (FIG. 9B) (ABE-275, amino acid sequence denoted by SEQ ID NO: 210; ABE-274, amino acid sequence denoted by SEQ ID NO: 204; ABE-254, amino acid sequence denoted by SEQ ID NO: 347; ABE-245, amino acid sequence denoted by SEQ ID NO: 338). The total base substitutions over the spacer window with guides targeting the AAVS1 locus are shown for the indicated ABEs.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0024] The Sequence Listing filed herewith provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions and systems according to the disclosure. Below are exemplary descriptions of sequences therein.

[0025] SEQ ID NO: 1 shows the protein sequence of MG3-6 3-8 nickase.

[0026] SEQ ID NOs: 2-8 show the nucleic acid sequences of ABE cds for monomeric MG68-4 ADA screen

[0027] SEQ ID NO: 9 shows the nucleic acid sequence of MG3-6_3-8 sgRNA targeting mouse Apoal .- 7 -#137172Attomey Docket No : 00010.037.1801

[0028] SEQ ID NOs: 10-32 show the nucleic acid sequences for Arginine-scanning.

[0029] SEQ ID NOs: 33-118 show the nucleic acid sequences of the 2nd generation.

[0030] SEQ ID NOs: 119-179 show the nucleic acid sequences of the 3rd generation.

[0031] SEQ ID NO: 180 shows the nucleic acid sequence of MG34-29 sgRNA targeting human AAVS 1.

[0032] SEQ ID NO: 181 shows the nucleic acid sequence of MG102-71 sgRNA targeting human AAVS1.

[0033] SEQ ID NOs: 182-188 show the protein sequences of ABE cds for monomeric MG68-4 ADA screen.

[0034] SEQ ID NOs: 189-211 show the protein sequences of SMART Arginine-scanning.

[0035] SEQ ID NOs: 212-297 show the protein sequences of SMART ABE 2nd generation.

[0036] SEQ ID NOs: 298-358 show the protein sequences of SMART ABE 3rd generation.

[0037] SEQ ID NO: 359 shows the nucleic acid sequence of MG3-6_3-8 spacer targeting mouseApoal .

[0038] SEQ ID NO: 360 shows the nucleic acid sequence of MG34-29 spacer targeting human AAVS1.

[0039] SEQ ID NO: 361 shows the nucleic acid sequence of MG102-71 spacer targeting human AAVS1.

[0040] SEQ ID NOs: 362-369 show the full-length peptide sequences of MG nickases suitable for the engineered nucleic acid editing systems described herein. SEQ ID NO: 369 shows the sequence of an MG34 nickase suitable for the engineered nucleic acid editing systems described herein.

[0041] SEQ ID NO: 370 shows the full-length peptide sequence of Fam72A.

[0042] SEQ ID NOs: 371-375 show the sequences of uracil DNA glycosylase inhibitors suitable for the engineered nucleic acid editing systems described herein.

[0043] SEQ ID NOs: 376-382 and SEQ ID NOs: 429-434 show exemplary linker sequences for deaminase systems described herein.

[0044] SEQ ID NOs: 383-428 show the amino acid sequences of nuclear localization signals (NLS).

[0045] SEQ ID NOs: 435-440 show the full-length nucleotide sequences of AAVS 1 target sites.

[0046] SEQ ID NO: 441 shows the full-length nucleotide sequence of an hApoAl_l guide RNA.

[0047] SEQ ID NO: 442 shows the full-length nucleotide sequence of the hApoAl target site.- 8 -#137172Attomey Docket No : 00010.037.1801DETAILED DESCRIPTION

[0048] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0049] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology7, genomics and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M J. MacPherson, B.D. Flames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0050] As used herein, the singular forms "a”. "an" and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0051] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1 % of a given value.

[0052] The term “nucleotide,” as used herein, refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotides are monomeric units of a nucleic acid sequence e.g, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [aS]dATP, 7-deaza- dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of- 9 -#137172Attorney Docket No : 00010.037.1801 ddNTPs include, but are not limited to, ddATP, ddCTP. ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g, fluorophores) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels of nucleotides include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine. 6- carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X- rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP. [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dRl 10]ddCTP, [dTAMRA] ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP. FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein- 15 -dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770- 9-dATP, Fluorescein-12-ddUTP, Fluorescein- 12-UTP, and Fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY- FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY- TR-14-UTP. BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP. fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5 -UTP, Rhodamine Green-5 -dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically-modified nucleotide is biotin-dNTP. Non-limiting examples of biotinylated dNTPs include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-d ATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin- 16-dUTP, biotin-20-dUTP).

[0053] The terms "polynucleotide." “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary polynucleotides include, but are not limited to. DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), shorthairpin RNA (shRNA), micro-RNA (miRNA), ribozymes. cDNA, recombinant polynucleotides,- 10 -#137172Attorney Docket No : 00010.037.1801 branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA). nucleic acid probes, and primers. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. A polynucleotide can be exogenous or endogenous to a cell and / or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer. Non-limiting examples of modifications include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The sequence of nucleotides may be interrupted by nonnucleotide components.

[0054] The terms "peptide." “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertiary structure (e.g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, refer to natural and non-natural amino acids, including, but not limited to, modified amino acids. Modified amino acids include amino acids that have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. The term “amino acid” includes both D-amino acids and L-amino acids.

[0055] As used herein, the “non-native” refers to a nucleic acid or polypeptide sequence that is non-naturally occurring. Non-native refers to a non-naturally occurring nucleic acid or polypeptide sequence that comprises modifications such as mutations, insertions, or deletions. The term non- native encompasses fusion nucleic acids or polypeptides that encodes or exhibits an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubi quitin ating activity, etc.) of the nucleic acid or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence includes those linked to a- 11 -#137172Attorney Docket No : 00010.037.1801 naturally -occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.

[0056] As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer to an arrangement of genetic elements, e.g, a promoter, an enhancer, a polyadenylation sequence, etc., wherein an operation (e.g., movement or activation) of a first genetic element has some effect on the second genetic element. The effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, a regulatory element, which may comprise promoter and / or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0057] A “functional fragment” of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence. A biological activity of a DNA sequence includes its ability to influence expression in a manner attributed to the full-length sequence.

[0058] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring. An engineered peptide has. but does not require, low sequence identity (e.g, less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein. For example, VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its ammo acid sequence to a sequence that does not exist in nature; an engineered protein acquiring anew function or property. An “engineered” system comprises at least one engineered component.

[0059] The term “tracrRNA” or “tracr sequence” means trans-activating CRISPR RNA. tracrRNA interacts with the CRISPR (cr) RNA to form guide (g) RNA in type II and subtype V-B CRISPR- Cas systems. If the tracrRNA is engineered, it may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or sequence similarity' to a wild type exemplary' tracrRNA sequence (e.g., a tracrRNA from . pyogenes, S. aureus). tracrRNA may refer- 12 -#137172Attorney Docket No : 00010.037.1801 to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. The term tracrRNA encompasses a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g.. a tracrRNA from S. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a tracrRNA sequence has at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g, a tracrRNA from S'. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. Type II tracrRNA sequences can be predicted on a genome sequence by identifying regions with complementarity to part of the repeat sequence in an adjacent CRISPR array.

[0060] As used herein, a ‘’guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid can include a crRNA or a tracrRNA or a combination of both. The term guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to specifically bind to the target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary to the guide nucleic acid is called noncomplementary strand. A guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.” A nucleic acid-targeting segment can include a sub-segment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.”

[0061] The term “sequence identity” or “percent identity ” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences- 13 -#137172Attorney Docket No : 00010.037.1801 include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11. extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW with parameters of ; the Smith- Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAFFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0062] As used herein, the term “RuvC III domain7’ refers to a third discontinuous segment of a RuvC endonuclease domain (the RuvC nuclease domain being comprised of three discontiguous segments, RuvC L RuvC II, and RuvC III). A RuvC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam HMM PF18541 for RuvC III).

[0063] As used herein, the term “HNH domain” refers to an endonuclease domain having characteristic histidine and asparagine residues. An HNH domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0064] As used herein, the term “base editor” refers to an enzyme that catalyzes the conversion of one target base or base pair into another (e.g., A:T to G:C, C:G to T:A) without requiring the creation and repair of a double-strand break. An exemplary base editor is a deaminase. In some embodiments, the base editor comprises a deaminase and a nuclease that is deficient in nuclease activity7. In some embodiments, the base editor comprises a deaminase and a catalytically inactive nuclease. In some embodiments, the base editor comprises a fusion of a deaminase and a catalytically inactive nuclease.

[0065] As used herein, the term “deaminase” refers to a protein or enzyme that catalyzes a deamination reaction (i.e., a reaction that removes an amino group). Deaminases include adenosine deaminases, which catalyze the hydrolytic deamination of adenine or adenosine (e.g., an engineered adenosine deaminase that deaminates adenosine in DNA), and cytidine (or cytosine) deaminases, which catalyze the hydrolytic deamination of cytidine (or cytosine) or deoxy cytidine to uridine (or uracil) or deoxyuridine, respectively. The deaminase or deaminase domain can be a naturally-occurring deaminase or deaminase domain from an organism, such as a human,- 14 -#137172Attorney Docket No : 00010.037.1801 chimpanzee, gorilla, monkey, cow, dog, rat, mouse, or bacterium (e.g., E. coli), a variant of a naturally-occurring deaminase or deaminase domain, or a non-naturally occurring deaminase or deaminase domain.

[0066] The term “optimally aligned” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that have been aligned to maximal correspondence of amino acids residues or nucleotides, for example, as determined by the alignment producing a highest or “optimized” percent identity score.

[0067] As used herein, the term “complex” refers to a joining of at least two components. The two components may each retain the properties / activities they had prior to forming the complex or gain properties as a result of forming the complex. The joining includes, but is not limited to. covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion, or any other suitable method. Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof. For example, a complex comprises an endonuclease and a guide polynucleotide.

[0068] Included in the current disclosure are variants of any of the enzymes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conserv ative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%. at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the endonuclease protein sequences described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of one or more critical active site residues or guide RNA binding residues of the endonuclease are not disrupted.

[0069] Also included in the current disclosure are variants of any of the enzymes described herein with substitution of one or more catalytic residues to decrease or eliminate activity of the enzy me- 15 -#137172Attorney Docket No : 00010.037.1801(e.g., decreased-activity variants). In some embodiments, a decreased activity variant as a protein described herein comprises a disrupting substitution of at least one. at least two, or all three catalytic residues. In some embodiments, any of the endonucleases described herein can comprise a nickase mutation. In some embodiments, any of the endonucleases described herein can comprise a RuvC domain lacking nuclease activity. In some embodiments, any of the endonucleases described herein can be configured to cleave one strand of a double-stranded target deoxyribonucleic acid. In some embodiments, any of the endonucleases described herein can comprise can be configured to lack endonuclease activity or be catalytically dead.

[0070] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see. for e.g., Creighton. Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Try ptophan (W);7) Serine (S). Threonine (T); and8) Cysteine (C), Methionine (M).Overview

[0071] The discovery of new CRISPR enzymes with unique functionality and structure may offer the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving speed, specificity7, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, comparatively few functionally characterized CRISPR enzymes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR systems documented and speed the discovery of new oligonucleotide editing functionalities. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX / CasY CRISPR systems from metagenomic analysis of natural microbial communities.- 16 -#137172Attorney Docket No : 00010.037.1801

[0072] CRISPR systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. In their natural context, CRISPR systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30-40bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory' proteins / enzymes. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer- adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR systems are commonly organized into 2 classes, 5 types and 16 subty pes based on shared functional characteristics and evolutionary' similarity' (see FIG. 1).

[0073] Class 1 CRISPR systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.

[0074] Type I CRISPR systems are considered of moderate complexity in terms of components. In Type I CRISPR systems, the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex called Cascade, which also comprises a nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Type I nucleases function primarily as DNA nucleases.

[0075] Type III CRISPR systems may be characterized by the presence of a central nuclease, known as CaslO, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits. Like in Type I systems, the mature crRNA is processed from a pre-crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).

[0076] Type IV CRISPR systems possess an effector complex that comprises a highly reduced large subunit nuclease (csfl), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.

[0077] Class 2 CRISPR systems generally have single-poly peptide multidomain nuclease effectors, and comprise Types II, V and VI.- 17 -#137172Attorney Docket No : 00010.037.1801

[0078] Type II CRISPR systems are considered the simplest in terms of components. In Type II CRISPR systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Type II nucleases are known as DNA nucleases. Type II effectors generally exhibit a structure comprising a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary’) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

[0079] Type V CRISPR systems are characterized by a nuclease effector (e.g., Casl2) structure similar to that of Type II effectors, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR systems, Type V CRISPR systems are again known as DNA nucleases. Unlike Type II CRISPR systems, some Type V enzymes (e.g., Casl2a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.

[0080] Type VI CRISPR systems have RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (e.g., Casl3) comprises tw o HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also may not require a tracrRNA in some instances for processing of pre-crRNA into crRNA. Similar to type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.

[0081] Because of their simpler architecture, Class 2 CRISPR have been most widely adopted for engineering and development as designer nuclease / genome editing applications.

[0082] One of the early adaptations of such a system for in vitro use involved (i) recombinantly- expressed, purified full-length Cas9 (e.g., a Class 2, Type II Cas enzyme) isolated from S'. pyogenes SF370. (ii) purified mature ~42 nt crRNA bearing a ~20 nt 5 ’ sequence complementary to the target DNA sequence desired to be cleaved followed by a 3’ tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carry ing a T7 promoter sequence); (iii)- 18 -#137172Attorney Docket No : 00010.037.1801 purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg2+. A later improved, engineered system involved the crRNA of (ii) joined to the 5’ end of (iii) by a linker (e.g, GAAA) to form a single fused synthetic guide RNA (sgRNA) capable of directing Cas9 to a target by itself.

[0083] Such engineered systems can be adapted for use in mammalian cells by providing DNA vectors encoding (i) an ORF encoding codon-optimized Cas9 (e.g, a Class 2, Type II Cas enzyme) under a suitable mammalian promoter with a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal); and (ii) an ORF encoding an sgRNA (having a 5’ sequence beginning with G followed by 20 nt of a complementary7targeting nucleic acid sequence joined to a 3’ tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g., the U6 promoter).Base editing

[0084] Base editing is the conversion of one target base or base pair into another (e.g., A:T to G:C, C:G to T:A) without requiring the creation and repair of a double-strand break. The base editing may be achieved with the help of DNA and RNA base editors that allow the introduction of point mutations at specific sites, in either DNA or RNA. Generally, DNA base editors may comprise a fusion of a catalytically inactive nuclease and a catalytically active base-modification enzyme that acts on single-stranded DNAs (ssDNAs). RNA base editors may comprise of similar, RNA- specific enzymes. Base editing may increase the efficiency of gene modification, while reducing the off-target and random mutations in the DNA.

[0085] DNA base editors are engineered ribonucleoprotein complexes that act as tools for single base substitution in cells and organism. They may be created by fusing an engineered basemodification enzyme and a catalytically deficient CRISPR endonuclease variant that cannot cut dsDNA, but it is able to unfold the dsDNA in a protospacer adjacent motif (PAM) sequencedependent manner, such that a guide RNA can find its complementary target to indicate a ssDNA scission site. The guide RNA anneals to the complementary DNA, displacing a fragment of ssDNA and directing the CRISPR ‘scissors' to the base modification site. The cellular repair machinery will repair the nicked non-edited strand using information from the complementary edited template.

[0086] So far, two ty pes of DNA editors, cytosine base (CBEs) and adenine base editors (ABEs) have been developed. However, recent findings indicate that off-target modifications are present in DNA, and that many off-target modifications are also introduced into RNA by DNA base editors.- 19 -#137172Attomey Docket No : 00010.037.1801MG Base Editors

[0087] Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor; (b) an endonuclease configured to bind the base editor and is deficient in nuclease activity; and (c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-358; and (b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence.

[0088] In some embodiments, the base editor comprises a sequence with at least 80%. at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 75% identity7to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 182- 358. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-358. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 182-358.

[0089] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about- 20 -#137172Attorney Docket No : 00010.037.1801 70% identity to any one of SEQ IDNOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 182- 188. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 98% identity7to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-188. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 182-188.

[0090] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 189-21 1. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 75% identity' to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 189- 211. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 189-211. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 189-211.- 21 -#137172Attomey Docket No : 00010.037.1801

[0091] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%. at least 83%. at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 212- 297. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 98% identity' to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 212-297. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 212-297.

[0092] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 85% identity' to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 298- 358. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs:- 22 -#137172Attorney Docket No : 00010.037.1801 298-358. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 298-358. In some embodiments, the base editor comprises a sequence having 100% identity7to any one of SEQ ID NOs: 298-358.

[0093] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity- to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 2-8.

[0094] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor comprises a sequence having at least about 75% identity- to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the- 23 -#137172Attomey Docket No : 00010.037.1801 base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 2-8 and 1 fl- 179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 2-8 and 10-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 2-8 and 1 fl- 179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 2-8 and 10-179.

[0095] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 2-8. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 2-8. In some embodiments,- 24 -#137172Attorney Docket No : 00010.037.1801 the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 2-8.

[0096] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 10-32. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 10-32.

[0097] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity' to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 33- 1 18. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor- 25 -#137172Attomey Docket No : 00010.037.1801 is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity' to any one of SEQ ID NOs: 33-118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 33-118.

[0098] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 1 19-179. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity7to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ IDNOs: 119-179. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity' to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 119-179. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 1 19-179.- 26 -#137172Attorney Docket No : 00010.037.1801102-2

[0099] In some embodiments, the base editor comprises a sequence with at least 80%. at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 267, 275, and 292. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 267, 275, and 292.

[0100] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%. at least 75%. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity' to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base- 27 -#137172Attorney Docket No : 00010.037.1801 editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 88. 96. and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 88, 96, and 113. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 88. 96, and 113.102-66

[0101] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 70% identity7to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 270, 279, 287. and 295. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 97% identity7to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 270, 279, 287, and 295. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 270, 279. 287, and 295.

[0102] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least- 28 -#137172Attomey Docket No : 00010.037.1801 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity7to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 91, 100, 108, and 1 16. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 91. 100, 108. and 116. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 91 , 100, 108, and 1 16. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 91, 100, 108, and 116. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 91. 100, 108, and 116. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 91, 100, 108, and 116.102-68

[0103] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%. at least 83%. at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 271, 280. 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 80% identity' to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 271 , 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 95% identity to- 29 -#137172Attorney Docket No : 00010.037.1801 any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 271, 280, 288. and 296. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 271, 280, 288, and 296. In some embodiments, the base editor comprises a sequence having 100% identify to any one of SEQ ID NOs: 271, 280, 288, and 296.

[0104] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%. 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 100% sequence identify to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identify to any one of SEQ ID NOs: 92, 101, 109. and 117. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identify7to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identify to any one of SEQ ID NOs: 92. 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identify to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identify to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identify to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identify7to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identify to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identify to any one of SEQ ID NOs: 92, 101, 109, and 117. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 92, 101, 109, and 117.102-71

[0105] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at- 30 -#137172Attorney Docket No : 00010.037.1801 least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 75% identity to anyone of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 272. 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 272, 281 , 289, 350, and 351 . In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 272, 281. 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 272, 281, 289, 350, and 351. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 272, 281. 289, 350. and 351.

[0106] In some embodiments, the base editor is encoded by' a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%. 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 100% sequence identity to any one of SEQ ID NOs: 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 93. 102, 110, 171, and 172. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 93, 102, 110, 171, and 172. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 93, 102, 110, 171. and 172. In some embodiments, the base editor is encoded by- a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ- 31 -#137172Attorney Docket No : 00010.037.1801 ID NOs: 93, 102. 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 93, 102. 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 93, 102, 110. 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 93, 102, 110, 171, and 172. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity7to any one of SEQ ID NOs: 93, 102, 110, 171. and 172.102-79

[0107] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 70% identity7to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 274, 283, 291. and 297. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 97% identity7to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 274, 283, 291, and 297. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 274, 283. 291, and 297.

[0108] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least- 32 -#137172Attorney Docket No : 00010.037.1801 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity7to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 95, 104, 112, and 1 18. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 95. 104, 112. and 118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 95, 104, 1 12, and 1 18. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 95, 104, 112, and 118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 95. 104, 112, and 118. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 95, 104, 112, and 118.102-39

[0109] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%. at least 83%. at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 213, 268, 276, 277. 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 213. 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In- 33 -#137172Attorney Docket No : 00010.037.1801 some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 213, 268. 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having at least about 99% identity' to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 213, 268, 276, 277, 284, 285, and 293.

[0110] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105. 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity' to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 34, 89, 97. 98. 105. 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity' to any one of SEQ ID NOs: 34. 89. 97. 98, 105, 106. and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity' to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 34, 89, 97, 98, 105, 106, and 114.- 34 -#137172Attorney Docket No : 00010.037.1801102-53[OHl] In some embodiments, the base editor comprises a sequence with at least 80%. at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 269, 278. 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 269, 278, 286, and 294. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 269, 278, 286, and 294.

[0112] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%. at least 75%. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 90, 99, 107, and 1 15. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 90, 99, 107. and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 90, 99, 107, and- 35 -#137172Attorney Docket No : 00010.037.1801 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity' to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity' to any one of SEQ ID NOs: 90, 99, 107, and 115. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 90, 99, 107. and 1 15.34-29

[0113] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 216, 212. 229, 242, 255. and 331. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 95% identity7to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 216, 212, 229. 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 98% identity' to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 216, 212. 229, 242, 255. and 331. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 216, 212, 229, 242, 255, and 331.- 36 -#137172Attorney Docket No : 00010.037.1801

[0114] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%. at least 75%. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 33, 37, 50, 63, 76, and 152.34-38

[0115] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%. at least 83%. at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 217, 231, 244, and 257.- 37 -#137172Attorney Docket No : 00010.037.1801 In some embodiments, the base editor comprises a sequence having at least about 85% identity' to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 217, 231, 244. and 257. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 217, 231 , 244, and 257. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 217, 231, 244, and 257. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 217, 231 , 244, and 257.

[0116] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%. 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 100% sequence identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 38. 52, 65, and 78. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity' to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 38, 52, 65, and 78. In some embodiments, the base editor is encoded by a- 38 -#137172Attorney Docket No : 00010.037.1801 nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 38, 52, 65, and78.34-71

[0117] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 219, 233. 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 80% identity' to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 219, 233, 246. and 259. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 219, 233, 246, and 259. In some embodiments, the base editor comprises a sequence having 100% identity’ to any one of SEQ ID NOs: 219, 233. 246, and 259.

[0118] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity' to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 40. 54, 67, and 80. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity' to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity’ to any one of SEQ- 39 -#137172Attomey Docket No : 00010.037.1801 ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 40, 54, 67, and 80. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 40, 54, 67, and 80.34-72

[0119] In some embodiments, the base editor comprises a sequence with at least 80%. at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 220 and 260. In some embodiments, the base editor comprises a sequence having 100% identity' to any one of SEQ ID NOs: 220 and 260.- 40 -#137172Attorney Docket No : 00010.037.1801

[0120] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%. at least 75%. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity' to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 41 and 81. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 41 and 81.34- 78

[0121] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 70% identity' to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 225, 238, 251. and 265. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises- 41 -#137172Attomey Docket No : 00010.037.1801 a sequence having at least about 90% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 97% identity' to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 225, 238, 251, and 265. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 225, 238. 251, and 265.

[0122] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity7to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 46. 59. 72. and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity' to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity' to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 46, 59, 72, and 86. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 46, 59, 72, and 86.- 42 -#137172Attorney Docket No : 00010.037.180134-35

[0123] In some embodiments, the base editor comprises a sequence with at least 80%. at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 230, 243, and 256. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 230, 243, and 256.

[0124] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%. at least 75%. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity7to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base- 43 -#137172Attomey Docket No : 00010.037.1801 editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 51, 64, and 77. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 51, 64, and 77.

[0125] In some embodiments, the base editor comprises a deaminase. In some embodiments, the deaminase binds non-covalently to the endonuclease. In some embodiments, the deaminase is covalently linked to the endonuclease. In some embodiments, the deaminase is fused to the endonuclease.Endonucleases

[0126] Described herein, in certain embodiments, are endonucleases deficient in nuclease activity. In some embodiments, the endonuclease comprises RuvC domain and an HNH domain. In some embodiments, the RuvC domain lacks nuclease activity. In some embodiments, the endonuclease comprises a nickase mutation. In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease is a class 2, type II endonuclease. In some embodiments, the endonuclease is configured to cleave one strand of a target nucleic acid (e.g., DNA).

[0127] In some embodiments, the endonuclease is not a Cas9 endonuclease, a Casl4 endonuclease, a Casl2a endonuclease, a Casl2b endonuclease, a Cas 12c endonuclease, a Casl2d endonuclease, a Casl2e endonuclease, a Casl3a endonuclease, a Casl3b endonuclease, a Casl3c endonuclease, or a Cas 13d endonuclease. In some embodiments, the endonuclease has less than 80% identity' to a Cas9 endonuclease.Guide Polynucleotides

[0128] In some embodiments, the engineered system disclosed herein comprises an engineered guide polynucleotide, e.g. , a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.

[0129] In some embodiments, the engineered guide polynucleotide (e.g., engineered guide RNA) is configured to form a complex with the engineered endonuclease. In some embodiments, the engineered guide polynucleotide comprises a spacer sequence. In some embodiments, the spacer- 44 -#137172Attorney Docket No : 00010.037.1801 sequence is configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0130] In some embodiments, the guide polynucleotide comprises a sequence is encoded by a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%. at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity7to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity' to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity' to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity7to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any' one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 180 and 181.

[0131] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to any one of SEQ ID NOs: 180 and 181 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity' to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 90% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a- 45 -#137172Attomey Docket No : 00010.037.1801 sequence having at least about 96% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary7to a sequence having at least about 98% identity7to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 180 and 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary7to a sequence having 100% identity7to any one of SEQ ID NOs: 180 and 181.

[0132] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80%. 85%. 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 180 and 181.

[0133] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 85% sequence identity7to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181.

[0134] In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary7to a fungal genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.

[0135] In some embodiments, the guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the guide polynucleotide is 42-44 nucleotides in length. In some embodiments, the guide polynucleotide is 42 nucleotides in length. In some embodiments, the guide polynucleotide is 43 nucleotides in length. In some embodiments, the guide polynucleotide is 44 nucleotides in- 46 -#137172Attorney Docket No : 00010.037.1801 length. In some embodiments, the guide polynucleotide is 85-245 nucleotides in length. In some embodiments, the guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the guide RNA is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length. In some embodiments, the guide RNA is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70. about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240. about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides.

[0136] In some embodiments, the engineered guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the engineered guide polynucleotide comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments, all of the inter-nucleoside linkers of the engineered guide polynucleotide, or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.

[0137] In some embodiments, the engineered guide polynucleotide comprises modifications to a ribose sugar or nucleobase. In some embodiments, the engineered guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g, locked nucleic acids (LNA)), or an unlinked nbose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a nonsugar moiety, for example peptide nucleic acids (PNA) or morpholino nucleic acids.

[0138] In some embodiments, the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modifications comprise modifications made by- 47 -#137172Attorney Docket No : 00010.037.1801 altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2’, 3’, 4’, or 5’ positions, or combinations thereof. In some embodiments, nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides. A 2’ sugar modified nucleoside, in some embodiments, is a nucleoside that has a substituent other than -H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’-4’ biradical bridged) nucleosides. Examples of 2 ’-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl-RNA, 2’-O- methyl-RNA, 2’ -alkoxy -RNA, 2’-O-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro- RNA, and 2’-F-ANA nucleosides. In some embodiments, the modification in the ribose group comprises a modification at the 2’ position of the ribose group. In some embodiments, the modification at the 2’ position of the ribose group is selected from the group consisting of 2’-O- methyl, 2’-fluoro, 2’-deoxy, and 2’-O-(2-methoxyethyl).

[0139] In some embodiments, the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, the engineered guide polynucleotide comprises only modified sugars. In certain embodiments, the engineered guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a2’-O-methoxyethyl group. In some embodiments, the engineered guide polynucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications.

[0140] In some embodiments, the engineered guide polynucleotide comprises a hairpin comprising at least 8 base-paired ribonucleotides. In some embodiments, the engineered guide polynucleotide comprises a hairpin comprising at least 9 base-paired ribonucleotides. In some embodiments, the engineered guide polynucleotide comprises a hairpin comprising at least 10 base-paired ribonucleotides. In some embodiments, the engineered guide polynucleotide comprises a hairpin comprising at least 11 base-paired ribonucleotides. In some embodiments, the engineered guide polynucleotide comprises a hairpin comprising at least 12 base-paired ribonucleotides.

[0141] In some embodiments, the engineered guide polynucleotide comprises a DNA-targeting segment. In some embodiments, the DNA-targeting segment comprises a nucleotide sequence that is complementary to a target sequence. In some embodiments, the target sequence is in a target DNA molecule. In some embodiments, the engineered guide polynucleotide comprises a proteinbinding segment. In some embodiments, the protein-binding segment comprises two complementary’ stretches of nucleotides. In some embodiments, the two complementary stretches of nucleotides hybridize to form a double-stranded RNA (dsRNA) duplex. In some embodiments,- 48 -#137172Attomey Docket No : 00010.037.1801 the two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides.Base Editing Systems

[0142] Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor; (b) an endonuclease configured to bind the base editor and is deficient in nuclease activity; and (c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0143] Described herein, in certain embodiments, are engineered base editing systems comprising an engineered base editing system comprising: a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182- 358; and an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence.

[0144] In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 98% identity to any one of SEQ ID NOs:- 49 -#137172Attorney Docket No : 00010.037.1801 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising 100% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease; and c) an engineered guide polynucleotide.

[0145] In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 80% identity' to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 95% identity' to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide- 50 -#137172Attorney Docket No : 00010.037.1801 configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 98% identity7to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising 100% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0146] In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 75% identity7to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide- 51 -#137172Attorney Docket No : 00010.037.1801 polynucleotide comprising a sequence having at least about 80% identity' to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 90% identity' to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 90% identity' to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 95% identity' to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 97% identity' to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 97% identity7to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 98% identity' to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 98% identity7to SEQ ID NO: 180 or- 52 -#137172Attorney Docket No : 00010.037.1801SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising 100% identity to any one of SEQ ID NOs: 182-358; b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising 100% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to SEQ ID NO: 180 or SEQ ID NO: 181 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181.

[0147] Described herein, in certain embodiments, are engineered base editing systems comprising an engineered base editing system comprising: a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity’ to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213. 268, 276, 277. 284, 285. and 293; SEQ ID Nos: 269, 278. 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); and an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence.

[0148] In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 70% identity' to any one of SEQ ID NOs: 182-358 (e.g.. SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225. 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 75% identity’ to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and- 53 -#137172Attorney Docket No : 00010.037.1801 292; SEQ ID NOs: 270, 279, 287. and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213, 268. 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 80% identity' to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos:269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229. 242, 255, and 331; SEQ ID Nos: 217. 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242. 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219, 233. 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238. 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide poly ucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288. and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238. 251, and 265; SEQ ID Nos: 230. 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 95% identity’ to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs:270, 279. 287, and 295; SEQ ID NOs: 271, 280, 288. and 296; SEQ ID Nos: 272, 281, 289. 350, and 351; SEQ ID Nos: 274. 283, 291, and 297: SEQ ID Nos: 213, 268. 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 21 , 212, 229, 242, 255, and 331 ; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260;- 54 -#137172Attorney Docket No : 00010.037.1801 SEQ ID Nos: 225, 238, 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281. 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268. 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278. 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216. 212, 229, 242. 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287. and 295; SEQ ID NOs: 271. 280, 288. and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238. 251, and 265; SEQ ID Nos: 230. 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279. 287, and 295; SEQ ID NOs: 271, 280, 288. and 296; SEQ ID Nos: 272, 281, 289. 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide. In some embodiments, the engineered system comprises a) a base editor comprising 100% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296;- 55 -#137172Attorney Docket No : 00010.037.1801 SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284. 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331 ; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease; and c) an engineered guide polynucleotide.

[0149] In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255. and 331; SEQ ID Nos: 217. 231, 244, and 257; SEQ ID Nos: 219. 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251 , and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs:270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297: SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278. 286, and 294; SEQ ID Nos: 216, 212, 229. 242, 255. and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295: SEQ ID NOs:271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at- 56 -#137172Attorney Docket No : 00010.037.1801 least about 85% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287. and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351 ; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275. and 292; SEQ ID NOs: 270, 279. 287, and 295; SEQ ID NOs: 271 , 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351 ; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos:269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217. 231, 244, and 257; SEQ ID Nos: 219. 233, 246. and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 182-358 (e.g.. SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255. and 331; SEQ ID Nos: 217. 231, 244, and 257; SEQ ID Nos: 219. 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 96% identity7to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs:270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278. 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255. and 331; SEQ ID Nos: 217, 231 , 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease- 57 -#137172Attorney Docket No : 00010.037.1801 configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295: SEQ ID NOs:271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287. and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos:272, 281, 289, 350, and 351 ; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275. and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351 ; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219. 233, 246. and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the engineered system comprises a) a base editor comprising 100% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275. and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284,- 58 -#137172Attorney Docket No : 00010.037.1801 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219. 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence.

[0150] In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291. and 297; SEQ ID Nos: 213, 268. 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278. 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331 ; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 182-358 (e.g.. SEQ ID NOs: 267. 275, and 292; SEQ ID NOs: 270, 279. 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219, 233. 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238. 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213. 268, 276, 277. 284, 285. and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331 ; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265;- 59 -#137172Attorney Docket No : 00010.037.1801 SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 85% identity’ to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212. 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219. 233, 246. and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 90% identity to any' one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213. 268, 276, 277. 284, 285. and 293; SEQ ID Nos: 269, 278. 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 95% identity’ to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212. 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219. 233, 246. and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and- 60 -#137172Attorney Docket No : 00010.037.1801 comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295: SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285. and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212. 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219. 233, 246. and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225. 238, 251. and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295: SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to SEQ- 61 -#137172Attorney Docket No : 00010.037.1801ID NO: 180 or SEQ ID NO: 181. In some embodiments, the engineered system comprises a) a base editor comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 182- 358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272, 281, 289, 350, and 351; SEQ ID Nos: 274, 283, 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285. and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212. 229, 242, 255. and 331; SEQ ID Nos: 217, 231, 244. and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to SEQ ID NO: 180 or SEQ ID NO: 181 . In some embodiments, the engineered system comprises a) a base editor comprising 100% identity to any one of SEQ ID NOs: 182-358 (e.g., SEQ ID NOs: 267, 275, and 292; SEQ ID NOs: 270, 279, 287, and 295; SEQ ID NOs: 271, 280, 288, and 296; SEQ ID Nos: 272. 281, 289, 350. and 351; SEQ ID Nos: 274, 283. 291, and 297; SEQ ID Nos: 213, 268, 276, 277, 284, 285, and 293; SEQ ID Nos: 269, 278, 286, and 294; SEQ ID Nos: 216, 212, 229, 242, 255, and 331; SEQ ID Nos: 217, 231, 244, and 257; SEQ ID Nos: 219, 233, 246, and 259; SEQ ID Nos: 220 and 260; SEQ ID Nos: 225, 238, 251, and 265; SEQ ID Nos: 230, 243, and 256); b) an endonuclease configured to bind the base editor; and c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising 100% identity' to SEQ ID NO: 180 or SEQ ID NO: 181. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to SEQ ID NO: 180 or SEQ ID NO: 181 or a sequence having at least 90%. 95%. 97%, 98%, or 99% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181.

[0151] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino acid sequence having about 85% sequence identity to the amino acid sequence of SEQ ID NO: 182. In some embodiments, the nickase domain comprises an ammo acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 182. In some embodiments, the nickase domain comprises an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 182. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 182.

[0152] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino- 62 -#137172Attorney Docket No : 00010.037.1801 acid sequence having about 85% sequence identity to the amino acid sequence of SEQ ID NO:183. In some embodiments, the nickase domain comprises an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 183. In some embodiments, the nickase domain comprises an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 183. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 183.

[0153] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino acid sequence having about 85% sequence identity to the amino acid sequence of SEQ ID NO:184. In some embodiments, the nickase domain comprises an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 184. In some embodiments, the nickase domain comprises an amino acid sequence having about 95% sequence identity7to the amino acid sequence of SEQ ID NO: 184. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 184.

[0154] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino acid sequence having about 85% sequence identity' to the amino acid sequence of SEQ ID NO:185. In some embodiments, the nickase domain comprises an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 185. In some embodiments, the nickase domain comprises an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 185. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 185.

[0155] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino acid sequence having about 85% sequence identity to the amino acid sequence of SEQ ID NO:186. In some embodiments, the nickase domain comprises an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 186. In some embodiments, the nickase domain comprises an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 186. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 186.

[0156] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino acid sequence having about 85% sequence identity to the amino acid sequence of SEQ ID NO:187. In some embodiments, the nickase domain comprises an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 187. In some embodiments, the- 63 -#137172Attorney Docket No : 00010.037.1801 nickase domain comprises an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 187. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 187.

[0157] In some embodiments, the endonuclease or base editor comprises one or more modifications in a nickase domain. In some embodiments, the nickase domain comprises an amino acid sequence having about 85% sequence identity to the amino acid sequence of SEQ ID NO: 188. In some embodiments, the nickase domain comprises an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 188. In some embodiments, the nickase domain comprises an amino acid sequence having about 95% sequence identity7to the amino acid sequence of SEQ ID NO: 188. In some embodiments, the nickase domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 188.

[0158] In some embodiments, the nickase domain comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 362, residue 13 relative to SEQ IDNOs: 363, 364, or 366, residue 12 relative to SEQ ID NO: 365, residue 17 relative to SEQ ID NO: 367, residue 23 relative to SEQ ID NO: 368. or residue 10 relative to SEQ ID NO: 369, or any combination thereof. In some embodiments, the endonuclease or base editor comprises a substitution of 109N and at least one other substitution comprising any one of 24R, 37L, 49A, 52L, 83S, 85F, 107V, 110S, 112R, 120N, 123N, 124Y, 147C, 148Y, 148R, 150Y, 153P, 154P, 155R, 156V, 157F, 158N, 1661, or 129N, or any combination thereof relative to SEQ ID NO: 1 when optimally aligned. In some embodiments, the endonuclease or base editor comprises at least one substitution of a wild-type amino acid for a non-wild-type amino acid comprising any7one of W90A, W90F, W90H, W90Y, Y120F, Y120H, Y121F, Y121H, Y121Q, Y121A, Y121D, Y121W, H122Y, H122F, H122I, H122A, H122W, H122D, Y121T, R33A, R34A, R34K, H122A, R33A, R34A, R52A, N57G, H122A, E123A, E123Q, W127F, W127H, W127Q. W127A, W127D, R39A. K40A, H128A, N63G, R58A, H121F, H121Y, H121Q, H121A, H121D, H121W, R33A, K34A, H122A, H121A, R52A, P26R, P26A, N27R, N27A, W44A, W45A, K49G, S50G, R51G, R121A, I122A, N123A, Y88F, Y120F, P22R, P22A, K23A, K41R. K41A, E54A, E54A, E55A, K30A, K30R, M32A, M32K, Y117A, K118A, I119A, I119H, R120A, R121A, P46A. P46R, N29A, R27A. orN50G, or any combination thereof. In some embodiments, the nickase comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 362, residue 13 relative to SEQ ID NOs: 363, 364, or 366, residue 12 relative to SEQ ID NO: 365, residue 17 relative to SEQ ID NO: 367, residue 23 relative to SEQ ID NO: 368, or residue 10 relative to SEQ ID NO: 369. or any combination thereof.

[0159] In some embodiments, the endonuclease or base editor comprises one or more modifications, e.g., arginine substitutions. In some embodiments, the endonuclease or base editor comprises one or more modifications selected from the group consisting of: S12R, T132R, Q158R,- 64 -#137172Attorney Docket No : 00010.037.1801 E277R, T278R, N372R, L598R, E672R, L696R, N701R, T378R, N745R, P820R, S185R, Q202R, W367R, Q657R. T681R, S697R, S748R, G846R. L828R. and P797R.

[0160] In some embodiments, the engineered system further comprises a uracil DNA glycosylase inhibitor. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 97% identity' to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 371-375. In some embodiments, the uracil DNA glycosylase inhibitor comprises a sequence having 100% identity to any one of SEQ ID NOs: 371-375.

[0161] In some embodiments, the base editor binds non-covalently to the endonuclease. In some embodiments, the base editor is covalently linked to the endonuclease. In some embodiments, the base editor is fused to the endonuclease at the N-terminus or at the C-terminus. In some embodiments, the base editor is fused to the endonuclease.

[0162] In some embodiments, the endonuclease is covalently coupled linked to the base editor or covalently linked to the base editor through a linker. In some embodiments, the linker comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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- 65 -#137172Attorney Docket No : 00010.037.1801 100% sequence identity to SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 429), SGSETPGTSESATPESA (SEQ ID NO: 430), GSGGS (SEQ ID NO: 431), SGSETPGTSESATPES (SEQ ID NO: 432), SGGSS (SEQ ID NO: 433), or GAAA (SEQ ID NO: 434). In some embodiments, the linker comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% sequence identity to any one of SEQ ID NOs: 376-382.Table 1: Linkers for deaminase systems described herein

[0163] In some embodiments, the system further comprises a source of Mg2+.

[0164] In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the endonuclease. In some embodiments, the base editor comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the endonuclease. The NLS can comprise any of the sequences in Table 2 below, or a combination thereof.

[0165] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 383- 428, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about35%. at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 85% identity7to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence- 66 -#137172Attorney Docket No : 00010.037.1801 having at least about 91% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 383- 428. In some embodiments, the NLS comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 97% identity7to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 383-428. In some embodiments, the NLS comprises a sequence having 100% identity to any one of SEQ ID NOs: 383-428.Table 2: Example NLS Sequences that can be used with Effectors According to the Disclosure- 67 -#137172Attorney Docket No : 00010.037.1801- 68 -#137172Attorney Docket No : 00010.037.1801Cells

[0166] Described herein, in certain embodiments, is a cell comprising the systems described herein.

[0167] In some embodiments, the cell is a eukaryotic cell (e.g, a plant cell, an animal cell, aprotist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NSO), or human retinal cells), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, aN2a cell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or aHeliothis virescens cell), a yeast cell (e.g, a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g. , a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g, a E. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.

[0168] In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell, or derivative thereof.

[0169] In some embodiments, the present disclosure provides a cell (e.g, host cell) comprising a vector described herein. In some embodiments, the cell expresses the engineered system described herein or components thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is genome edited ex vivo. In some embodiments, the cell is genome edited in vivo.

[0170] Described herein, in some embodiments, are host cells comprising an open reading frame encoding a heterologous endonuclease and a heterologous base editor having at least 75% sequence identity to any one of SEQ ID NOs: 1 and 182-358. In some embodiments, said heterologous base editor comprises a sequence having at least about 75%. at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at- 69 -#137172Attorney Docket No : 00010.037.1801 least about 96%, at least about 97%, at least about 98%. at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1 and 182-358.

[0171] In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is Bifidobacterium longum. Bifidobacterium lactis. Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum. Lactobacillus fermentum, Lactococcus lactis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus diacetylactis, Lactococcus cremoris, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii, or Escherichia coli. In some embodiments, the host cell is an E. coli cell. In some embodiments, the E. coli cell is a ZDE3 lysogen or a BL21(DE3) strain. In some embodiments, the E. coli cell has an ompT Ion genotype.

[0172] In some embodiments, the cell is within a cochlea. In some embodiments, the cell is within an embr o. In some embodiments, the embryo is a two-cell embry o. In some embodiments, the embryo is a mouse embryo.Lipid nanoparticles

[0173] Lipid nanoparticles as described herein can be 4-component lipid nanoparticles. Such nanoparticles can be configured for delivery’ of RNA or other nucleic acids (e.g. synthetic RNA, mRNA, or in vzfro-synthesized mRNA) and can be generally formulated as described in WO2012135805A2. Such nanoparticles can generally comprise: (a) a cationic lipid (e.g. 98N12-5 (TETA5-LAP), DLin DMA, DLin-K-DMA (2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane). DLin-KC2-DMA, DLin-MC3-DMA, or C 12-200). (b) a neutral lipid (e.g. DSPC or DOPE), (c) a sterol (e.g. cholesterol or a cholesterol analog), and (d) a PEG-modified lipid (e.g. PEG-DMG).

[0174] The cationic lipid referred to herein as “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670. Cationic lipid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or RNA (e.g. mRNA). As an example, formulations with certain cationic lipids include, but are not limited to, 98N12-5, and may contain 42% lipidoid, 48% cholesterol, and 10% PEG (Cl 4 or greater alkyl chain length). As another example, formulations with certain lipidoids include, but are not limited to, C 12-200 and may contain 50% cationic lipid, 10% disteroylphosphatidyl choline. 38.5% cholesterol, and 1.5% PEG-DMG.

[0175] In some embodiments, lipid nanoparticles are formulated as described in US10709779B2. In some embodiments, the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified- 70 -#137172Attorney Docket No : 00010.037.1801 lipid, a sterol, and a non-cationic lipid. In some embodiments, the cationic lipid is selected from the group consisting of 98N12-5 (TETA5-LAP). DLin DMA. DLin-K-DMA (2.2-Dilinoleyl-4- dimethylaminomethyl-[I,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA, and C 12-200. In some embodiments, the cationic lipid nanoparticle has a molar ratio of about 20-60% cationic lipid, about 5-25% non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 50% cationic lipid, about 1.5% PEG-modified lipid, about 38.5% cholesterol, and about 10% non-cationic lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 55% cationic lipid, about 2.5% PEG-modified lipid, about 32.5% cholesterol, and about 10% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, the cationic lipid nanoparticle has a molar ratio of 50:38.5: 10: 1.5 of cationic lipid: cholesterol: PEG2000-DMG:DSPC or DMG:DOPE. In some embodiments, lipid nanoparticles as described herein can comprise cholesterol, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,l ‘-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl)bis(dodecan-2-ol) (Cl 2-200), and DMG-PEG-2000 at molar ratios of 47.5: 16:35:1.5.Delivery and Vectors

[0176] Disclosed herein, in some embodiments, are nucleic acids encoding an engineered system described herein comprising a base editor, an endonuclease, and an engineered guide polynucleotide or components thereof (e.g., a base editor, an endonuclease, or an engineered guide polynucleotide).

[0177] In some embodiments, the nucleic acid encoding the engineered system or components thereof is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the engineered system is an RNA, for example a mRNA.

[0178] In some embodiments, the nucleic acid encoding the engineered system or components thereof is delivered by a nucleic acid-based vector. In some embodiments, the nucleic acid-based vector is a plasmid (e.g, circular DNA molecules that can autonomously replicate inside a cell), cosmid (e g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), Pl -derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus. In some embodiments, the nucleic acid-based vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT- 3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20- COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-- 71 -#137172Attorney Docket No : 00010.037.1801 daGFP. pEFla-mCherry-Nl vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF- CMV-PGK-Puro, pMCP-tag(m). pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal,pSF- OXB20-Fluc, pSF-OXB20, pSF-Tac, pRI 101-AN DNA, pCambia2301,pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0179] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, an open reading frame is operably linked to the promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, pl9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof. In some embodiments the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter.

[0180] In some embodiments, the open reading frame is operably linked to a T7 promoter sequence, a T7-lac promoter sequence, a lac promoter sequence, a tac promoter sequence, a trc promoter sequence, a ParaBAD promoter sequence, a PrhaBAD promoter sequence, a T5 promoter sequence, a cspA promoter sequence, an araPBAD promoter, a strong leftward promoter from phage lambda (pL promoter), or any combination thereof.

[0181] In some embodiments, the open reading frame comprises a sequence encoding an affinity tag linked in-frame to a sequence encoding said base editor. In some embodiments, the affinity tag is an immobilized metal affinity chromatography (IMAC) tag. In some embodiments, the IMAC tag is a polyhistidine tag. In some embodiments, the affinity tag is a myc tag, a human influenza hemagglutinin (HA) tag, a maltose binding protein (MBP) tag, a glutathione S-transferase (GST) tag, a streptavidin tag, a FLAG tag, or any combination thereof.

[0182] In some embodiments, the affinity tag is linked in-frame to said sequence encoding said base editor via a linker sequence encoding a protease cleavage site. In some embodiments, the protease cleavage site is a tobacco etch virus (TEV) protease cleavage site, a PreScission® protease (PSP) cleavage site, a Thrombin cleavage site, a Factor Xa cleavage site, an enterokinase cleavage site, or any combination thereof.

[0183] In some embodiments, the open reading frame is codon-optimized for expression in said host cell. In some embodiments, the open reading frame is provided on a vector. In some embodiments, the open reading frame is integrated into a genome of said host cell.

[0184] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In- 72 -#137172Attorney Docket No : 00010.037.1801 some embodiments, the virus is a baculovirus. In some embodiments, the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.

[0185] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV- rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV- Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV- LK03, AAV-HSC1, AAV-HSC2. AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV- HSC7, AAV-HSC8. AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC 14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV- NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8

[0186] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV 10 or a derivative thereof. In some embodiments, the virus is AAV 11 or a derivative thereof. In some embodiments, the virus is AAV 12 or a derivative thereof. In some embodiments, the virus is AAV 13 or a derivative thereof. In some embodiments, the virus is AAV 14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV 16 or a derivative thereof. In some embodiments, the vims is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO or a derivative thereof. In some embodiments, the vims is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV -rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the vims is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the vims is AAV-PHP-B or a derivative thereof. In some embodiments, the vims is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the vims- 73 -#137172Attorney Docket No : 00010.037.1801 is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the vims is AAV-HSC8 or a derivative thereof. In some embodiments, the vims is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV -HSC 12 or a derivative thereof. In some embodiments, the vims is AAV-HSC13 or a derivative thereof. In some embodiments, the vims is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the vims is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the vims is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0187] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the vims is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.

[0188] In some embodiments, the nucleic acid encoding the engineered system, the endonuclease, or the engineered guide polynucleotide is delivered by a non-nucleic acid-based delivery system (e.g, a non-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise- 74 -#137172Attorney Docket No : 00010.037.1801 associated with a lipid. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0189] In some embodiments, the engineered system, the endonuclease, or the engineered guide polynucleotide is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, the engineered system, the endonuclease, or the engineered guide polynucleotide is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes the engineered system, the endonuclease, or the engineered guide polynucleotide. For example, a cell is transduced (e.g., with a virus encoding the engineered system, the endonuclease, or the engineered guide polynucleotide), or transfected (e.g., with a plasmid encoding the engineered system, the endonuclease, or the engineered guide polynucleotide) with a nucleic acid that encodes the engineered system, the endonuclease, or the engineered guide polynucleotide, or the translated engineered system or endonuclease. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, cells expressing the engineered system, the endonuclease, or the engineered guide polynucleotide are transduced or transfected with one or more gRNA molecules. In some embodiments, a plasmid expressing the engineered system, the endonuclease, or the engineered guide polynucleotide is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g, piggybac) and viral transduction (for example lenti virus or AAV) or other methods known to those of skill in the art. In some embodiments, the engineered system, the endonuclease, or the engineered guide polynucleotide is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and / or RNPs are known in the art, for example by electroporation or by cell squeezing.

[0190] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, poly cation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91 / 17424 and WO 91 / 16024. In some embodiments, the delivery is to cells (e.g, in vitro or ex vivo administration) or target tissues (e.g.. in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.- 75 -#137172Attorney Docket No : 00010.037.1801

[0191] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.Methods of Use

[0192] Described herein, in certain embodiments, are methods of modifying a target nucleic acid with the base editor or engineered systems described herein, disrupting a gene locus using the base editor or engineered systems described herein, or manufacturing a base editor or engineered systems described herein.

[0193] In some embodiments, the engineered system comprises an adenine deaminase base editor, the nucleotide is an adenine, and modifying the target nucleic acid locus comprises converting the adenine to a guanine. In some embodiments, the engineered system comprises a cytidine deaminase base editor and a uracil DNA glycosylase inhibitor, the nucleotide is a cytosine, and modifying the target nucleic acid locus comprises converting the cytosine to a uracil.

[0194] In some embodiments, the methods are used to introduce a modification in the genome of a cell. In some embodiments, the target nucleic acid is modified in vitro. In some embodiments, the target nucleic acid sequence is modified in vivo. In some embodiments, the target nucleic acid sequence is modified ex vivo.

[0195] In some embodiments, the target nucleic acid comprises genomic DNA, viral DNA, or bacterial DNA. In some embodiments, the target nucleic acid is within a cell. In some embodiments, the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell. In some embodiments, the cell is within an animal.

[0196] In some embodiments, the target nucleic acid comprises DNA. In some embodiments, the DNA comprises a first strand comprising a sequence complementary to a sequence of the engineered guide polynucleotide and a second strand comprising a PAM. In some embodiments, the PAM is directly adjacent to the 3' end of the sequence complementary to the sequence of the engineered guide polynucleotide.

[0197] In some embodiments, the present disclosure provides a method of modifying a target nucleic acid (e.g., gene) locus. In some embodiments, the method comprises delivering to the target nucleic acid locus the engineered system described herein. In some embodiments, the endonuclease is configured to form a complex with the engineered guide polynucleotide. In some embodiments, the complex is configured such that upon binding of the complex to the target nucleic acid locus, the complex modifies the target nucleic acid locus.

[0198] In some embodiments, delivery of the engineered system to the target nucleic acid locus comprises delivering the nucleic acid described herein or the vector described herein. In some- 76 -#137172Attorney Docket No : 00010.037.1801 embodiments, delivery' of the engineered system to the target nucleic acid locus comprises delivering a nucleic acid comprising an open reading frame encoding the base editor and the endonuclease. In some embodiments, the nucleic acid comprises a promoter. In some embodiments, the open reading frame encoding the base editor and the endonuclease is operably linked to the promoter.

[0199] In some embodiments, delivery of the engineered system to the target nucleic acid locus comprises delivering a capped mRNA containing the open reading frame encoding the base editor and the endonuclease. In some embodiments, delivery of the engineered system to the target nucleic acid locus comprises delivering a translated polypeptide. In some embodiments, delivery of the engineered system to the target nucleic acid locus comprises delivering a deoxyribonucleic acid (DNA) encoding the engineered guide RNA operably linked to a ribonucleic acid (RNA) pol III promoter.

[0200] In some embodiments, the target gene is TRAC.

[0201] In some embodiments, the target gene is AAVS1. In some embodiments, the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 93% identity' to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 94% identity' to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about- 77 -#137172Attorney Docket No : 00010.037.1801 98% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to SEQ ID NO: 180 or 181. In some embodiments, the gRNA comprises a sequence having 100% identity to SEQ ID NO: 180 or 181.

[0202] In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%. at least about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% identity' to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 70% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 75% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 80% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS 1 sequence having at least about 85% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAV S 1 sequence having at least about 90% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 91% identity' to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 92% identity’ to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS 1 sequence having at least about 93% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 94% identity' to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 95% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 96% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 97% identity' to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having at least about 98% identity’ to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS 1 sequence having at least about 99% identity to any one of SEQ ID NOs: 435-440. In some embodiments, the gRNA hybridizes to a AAVS1 sequence having 100% identity' to any one of SEQ ID NOs: 435-440.

[0203] In some embodiments, the target gene is hApoAl. In some embodiments, the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least- 78 -#137172Attorney Docket No : 00010.037.1801 about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity' to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having atleast about 70% identity' to SEQ IDNOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 75% identity’ to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to SEQ ID NOs:441. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to SEQ ID NOs: 441 . In some embodiments, the gRNA comprises a sequence having at least about 93% identity' to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to SEQ ID NOs:441. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 96% identity' to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 97% identity' to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having at least about 99% identity' to SEQ ID NOs: 441. In some embodiments, the gRNA comprises a sequence having 100% identity' to SEQ ID NOs: 441.

[0204] In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 20%, at least about 25%. at least about 30%, at least about 35%, at least about 40%, at least about45%. at least about 50%. at least about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% identity' to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 70% identity to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 75% identity7to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 80% identity to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 85% identity to SEQ ID NO:442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 90% identity7to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 91% identity' to SEQ ID NO: 442. In some embodiments, the gRNA- 79 -#137172Attorney Docket No : 00010.037.1801 hybridizes to ahApoAl sequence having at least about 92% identity to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 93% identity to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 94% identity' to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 95% identity' to SEQ ID NOs: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 96% identity to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 97% identity' to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having at least about 98% identity' to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to ahApoAl sequence having at least about 99% identity to SEQ ID NO: 442. In some embodiments, the gRNA hybridizes to a hApoAl sequence having 100% identity to SEQ ID NO: 442.

[0205] Described herein, in certain embodiments, are methods of modilying a nucleic acid encoding APOA1 comprising contacting the nucleic acid sequence encoding APOA1 with an engineered base editing system, said base editing system comprising: a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-358.

[0206] In certain embodiments, disclosed herein are methods of modifying a nucleic acid encoding APOA1 comprising contacting the nucleic acid sequence encoding APOA1 with an engineered base editing system, said base editing system comprising: a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-358. wherein the sequence does not comprise any one of the sequences selected from SEQ ID NO: 362-369; and an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181.

[0207] In some embodiments, the endonuclease induces a single-stranded break or a doublestranded break at or proximal to the target locus. In some embodiments, the endonuclease induces a staggered single stranded break within or 5’ to said target locus. In some embodiments, the endonuclease does not induce a break at or proximal to the target locus.

[0208] In some embodiments, the present disclosure provides methods of manufacturing or producing a base editor. In some embodiments, the method comprises cultivating the cell. In some embodiments, the methods of producing a base editor, comprising cultivating the host cell described herein in compatible growth medium. In some embodiments, the methods further comprise inducing expression of said base editor by addition of an additional chemical agent or an- 80 -#137172Attorney Docket No : 00010.037.1801 increased amount of a nutrient. In some embodiments, the chemical agent is Isopropyl (3-D-l- thiogalactopyranoside (IPTG). In some embodiments, the nutrient is lactose. In some embodiments, the methods further comprise isolating said host cell after said cultivation and lysing said host cell to produce a protein extract. In some embodiments, the methods further comprise subjecting said protein extract to IMAC, or ion-affinity chromatography. In some embodiments, the methods further comprise cleaving said IMAC affinity tag by contacting a protease corresponding to said protease cleavage site to said base editor. In some embodiments, the methods further comprise performing subtractive IMAC affinity chromatography to remove said affinity tag from a composition comprising said base editor.

[0209] Systems of the present disclosure may be used for various applications, such as, for example, nucleic acid editing (e.g., gene editing), binding to a nucleic acid molecule (e.g, sequence-specific binding). Such systems may be used, for example, for addressing (e.g, removing or replacing) a genetically inherited mutation that may cause a disease in a subject, inactivating a gene in order to ascertain its function in a cell, as a diagnostic tool to detect disease-causing genetic elements (e.g. via cleavage of reverse-transcribed viral RNA or an amplified DNA sequence encoding a disease-causing mutation), as deactivated enzymes in combination with a probe to target and detect a specific nucleotide sequence (e.g., sequence encoding antibiotic resistance int bacteria), to render viruses inactive or incapable of infecting host cells by targeting viral genomes, to add genes or amend metabolic pathways to engineer organisms to produce valuable small molecules, macromolecules, or secondary metabolites, to establish a gene drive element for evolutionary selection, to detect cell perturbations by foreign small molecules and nucleotides as a biosensor.Kits

[0210] In some embodiments, this disclosure provides kits comprising one or more nucleic acid constructs encoding the various components of the engineered system described herein, e.g., comprising a nucleotide sequence encoding the components of the engineered editing system capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the engineered system components.

[0211] In some embodiments, any of the engineered editing systems disclosed herein is assembled into a pharmaceutical, diagnostic, or research kit to facilitate its use in therapeutic, diagnostic, or research applications. A kit may include one or more containers housing any of the vectors disclosed herein and instructions for use.

[0212] The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided- 81 -#137172Attorney Docket No : 00010.037.1801 in liquid form (e.g., in solution), or in solid form, (e.g, a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g. to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. 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, in some embodiments, are in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.EXAMPLES

[0213] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.Example 1 - Development of improved ADA variants that are active as a single open reading frame inlaid in a Type II nuclease

[0214] This Example describes generation of ADA variants.

[0215] Two SMART nuclease MG34-29 and MG102-71 (containing 748 aa and 943 aa, respectively) were used for engineering both the nickase and the ADA components. Domainwalking of the monomeric MG68-4 variant along the MG34-29 and MG102-71 nickase chassis was performed to identify the optimal insertion point for inlaid designs. Further rational engineering of the nickase identified SMART ABEs with robust A to G conversion activity demonstrated that SMART ABEs are active at two sites at the AAVS1 locus.

[0216] Methods for designing constructs

[0217] To generate pEditor plasmids, the study cloned ADA MG68-4 variants (with a combination of up to 7 mutations: V83S, L85F, D109N, T112R, R153P, R154P, A155R) at an L791 inlay site of a nickase chassis MG3-6_3-8 (SEQ ID NO: 1) via homology-based cloning and Type IIS restriction enzyme-based cloning. To generate a bacterial pReporter selection plasmid, the study cloned a chloramphenicol acetyltransferase mutant (C31Y, QI 15*) via the homology-based cloning. To generate a mammalian pReporter plasmid, the study cloned a stop codon-containing- 82 -#137172Attorney Docket No : 00010.037.1801 target spacer sequence (mApoAl) upstream of and in-frame with an mCherry reporter protein via Type IIS restriction enzyme-based cloning. DNA oligonucleotides and fragments were synthesized and sequencing verification was performed.

[0218] Methods for E. colt cell survival assay

[0219] The study induced expression of the gene editor and guide RNA using 0.2% arabinose, grown under selection conditions in E. coll cells (0 to 1280 pg / mL chloramphenicol), and quantified cell survival by calculating colony forming units (cfu) at each chloramphenicol concentration. Candidates were determined as sequences that imparted E. coll growth at chloramphenicol concentrations greater than E. coli cells expressing a reference enzy me and were selected to be tested in mammalian cells.

[0220] Methods for HEK293T cell fluorescence screen

[0221] Plasmids encoding the ADA candidates (SEQ ID NOs. 2-8) were tested using a mammalian fluorescence-based screen (FIG. 1A). A pEditor plasmid encodes ABE-T2A-GFP in which an ABE and GFP reporter are co-expressed and separated by a T2A ribosomal skipping peptide and a pReporter plasmid encodes BFP-T2A-target-mCherry that contains a target spacer sequence (FIG. IB). In cells, an ABE expressed from a pEditor edits an in-frame stop codon to a sense codon on a pReporter, and mCherry expression acts as an indicator of ABE activity. HEK293T cells were transfected using a transfection reagent (120 ng pEditor, 300 ng pReporter) and MessengerMAX (100 pmol synthetic gRNA). The study also measured the fraction of dually -transfected cells, which was the sub-population positive for both GFP and BFP. also expressing mCherry. This fraction of mCherry-positive cells in the dually-transfected sub-population is increased for ABE variants improving activity. Synthetic gRNAs containing phosphorothioate and 2'-O-methyl chemical modifications were also synthesized.

[0222] Results

[0223] Chloramphenicol selection andHEK293T cell fluorescence screen results for a single ADA orf inlaid ABE

[0224] Improved ADA MG68-4 variants that arose in the chloramphenicol selection exhibited improved activity in the HEK293T cell fluorescence screen (FIG. 1C). The study identified two improved adenosine deaminase variants providing five (5) orders of magnitude greater resistance to chloramphenicol and a 1.6-fold greater activity in the mammalian fluorescence-based screen. Variants 91 (nucleic acid sequence denoted by SEQ ID NO: 2 and amino acid sequence denoted by SEQ ID NO: 182) and 95 (nucleic acid sequence denoted by SEQ ID NO: 3 and amino acid sequence denoted by SEQ ID NO: 183) were used for subsequent protein engineering of SMART ABEs, specifically, domain-walking at an increased number of inlay sites.- 83 -#137172Attorney Docket No : 00010.037.1801Example 2 - Small ABEs engineered for higher editing in human cells

[0225] This Example describes use of small ABEs for editing in human cells.

[0226] Methods-SMART-nuclease derived small base editors

[0227] Set of ABEs for pooled screen - construct design

[0228] To generate pEditor plasmids, a single and a dual ORFs of ADA MG68-4 variants were inserted into unstructured loops and at both termini of SMART nickases (SEQ ID NOs: 33-118) via homology- based cloning and Type IIS restriction enzyme-based cloning. Unstructured loops of members from two SMART nuclease families (MG34, MG102) were identified via AlphaFold2 modeling and multiple sequence alignment. Clonal synthetic DNA encoding H. sapiens codon- optimized SMART nickases, with SapI cloning sites at the insertion points (inlaid at unstructured loops or at N- or C- terminal) and terminal Bsal sites, was procured and cloned into a polyA-less mRNA production vector using Bsal Golden Gate Assembly in a pooled manner. In addition, clonal synthetic DNA encoding single and dual ORF ADA MG68-4 with the terminal SapI sites was procured and cloned with SapI Golden Gate assembly into the SMART nickase chassis within the previously mentioned polyA-less mRNA production vector in a pooled manner. Sequence confirmation of the clonal isolates was performed of whole-construct colony PCR products.

[0229] mRNA production

[0230] Sequences for small ABEs were codon optimized for human expression and each was cloned into an expression vector with a T7 promoter, 5' and 3’ UTRs, and a poly A tail. The coding sequence comprised an N-terminal SV40 nuclear localization signal (NLS) and a C- terminal nucleoplasmin (NucP) nuclear localization signal. The expression vector was midiprepped, linearized with Spel, cleaned, and used for in vitro transcription with Hi-T7. In vitro transcription reactions contained N1 -methylpseudouridine in place of uridine and had added CleanCap reagent. The resulting mRNA was cleaned with RNeasy, checked for product size and purity and diluted to 250 ng / pL in sterile water for use in nucleofection.

[0231] Mammalian cell culture, transfections, and data analysis

[0232] K562 cells (ATCC CCL-243) were cultured in IMDM + GlutaMAX media and 10% FBS for 1-2 passages prior to the nucleofection. On the day of nucleofection, cells were harvested, counted, washed in IX PBS, and resuspended in the SF buffer. 120,000 cells were distributed per well and nucleofected with 500 ng of mRNA and 150 pmol-300 pmol of sgRNA. A second high- throughput pooled approach was developed by pooling between 2-5 mRNA ABE fusions with the same chassis each at 500 ng and tested across sixteen (16) nuclease validated guides at various loci. The general plate layout for this approach is shown in FIG. 2A and FIG. 2B. To identify which base editor in the pools contributed to editing, some base editors were tested individually at 500 ng. Cells were added to recovery' media and grown for 72 hours before genomic DNA was- 84 -#137172Attorney Docket No : 00010.037.1801 harvested. Resulting gDNA was diluted 1:3 and used as a template for NGS PCR. Targeted sequences were amplified with NGS primers. Amplicons around 250 bp long were checked, sequenced, and analyzed to measure gene editing outcomes.

[0233] Experimental Results-Pooled data and unpooled data

[0234] The 86 ABE variants which were designed (ABE115-ABE200, Tables 3-5) were split into 22 pools based off of the shared nickase chassis (e.g., a pool containing MG102-53 nickase with a C-terminal heterodimer, N-terminal heterodimer, inlaid monomer, or inlaid heterodimer deaminase). Each pool was tested with 16 guide RNAs for base editing activity depending on the PAM of the nickase and which guide scaffold the nickase preferred. From this screen, the study identified six pools with base editing activity >10% on at least one guide (FIG. 3). Unpooling was done for these six active pools and individual nucleofections were performed with each mRNA in the pool. Base editing conversion efficiencies up to 14% were observed for ABE-184 (SEQ ID NO: 350) and ABE-192 (SEQ ID NO: 289) in the MG102-71 pool with an NRC PAM (FIG. 4A and FIG. 4B), up to 30% for ABE-180 in the MG102-39 pool with an NAR PAM, and up to 30% for ABE- 158 in the MG34-29 pool with an NGG PAM (FIGs. 5A-5B and FIGs. 6A-6B).Table 3: Pooled mRNA approach, where each pool contains 3-4 variants with different MG68-4 fusions. All pools were tested with the same 16 MG102-2 guides- 85 -#137172Attorney Docket No : 00010.037.1801- 86 -#137172Attorney Docket No : 00010.037.1801Table 4: Pooled mRNA approach where each pool contains 3-4 variants with different MG68-4 fusions. All pools were tested with the same 16 MG102-29 guides- 87 -#137172Attorney Docket No : 00010.037.1801- 88 -#137172Attorney Docket No : 00010.037.1801Table 5: Pooled mRNA approach where each pool contains 2-5 variants with different MG68-4 fusions. All pools were tested with the same 16 MG34-1 guides- 89 -#137172Attorney Docket No : 00010.037.1801- 90 -#137172Attorney Docket No : 00010.037.1801- 91 -#137172Attorney Docket No : 00010.037.1801Example 3 - Structure-guided engineering of small ABEs improves editing in human cells

[0235] This Example describes structural-guided engineering of small ABEs

[0236] Domain walking of improved ADA MG68-4 in SMART nickase MG34-29 and MG 102-71

[0237] Methods for designing constructs, set of variants

[0238] To generate pEditor plasmids, the improved monomeric ADA MG68-4_Var91 (V83S, D109N, T112R, R154P) or MG68-4_Var95 (V83S, D109N, T112R, R153P, R154P) were inserted at unstructured loops of the MG34-29 (SEQ ID NOs. 119-152) and MG102-71 (SEQ ID NOs. 153- 172) nickases that were proximal to the substrate binding site. Unstructured loops were modeled in AlphaFold2 and unresolved in a cryo-EM structure. Specific inlay sites in MG102-71 were in the loop separating RuvC-II from EINH (D360), HNH domain (G389, K410, D426, L457), and RuvC-III (D563, N599, D621, E626). Specific inlay sites in MG34-29 were in the loop separating RuvC-I from REC lobe (D38), REC lobe (P102. E103, E104. K105, El 12, DI 14, N203). loop- 92 -#137172Attorney Docket No : 00010.037.1801 separating REC lobe from RuvC-II (E279), RuvC-III (S475, E491, K496. L516, E530); additional tested fusion sites were the N- and C-termini.

[0239] Arginine scan ofMG102-71 ABE

[0240] Methods for designing constructs, set of variants

[0241] Arginine substitutions were performed on MG102-71 variants that contained either an improved monomeric ADA MG68-4_Var91 (V83S, D109N, T112R, R154P) or MG68-4_Var95 (V83S, D109N, T112R, R153P, R154P) at the N599 inlay site (SEQ ID NOs. 10-32). Sites for mutagenesis were identified based on: (1) a lack of positively charged residues in MG102-71; (2) presence in Recognition Domain, WED domain, or Pam-interacting domain; (3) high frequency of R / K in other MG102 homologs; and (4) high solvent accessibility in the AlphaF old-predicted structure of MG102-71. A total of 23 positions were identified in MG102-71 to mutate to arginine and were introduced into either the MG68-4_Var91 or MG68-4_Var95 ADA constructs with the N599 inlay site (Table 6).Table 6: MG102-71 arginine mutations and associated ADA variant tested

[0242] Experimental Results

[0243] HEK293T cell fluorescence screen results for MG34-29 and MG102-71 domain walking. Based on the fraction of mCherry-positive cells in the dually -transfected sub-population, the study identified improved MG102-71 ABE variants (SEQ ID NOs: 153-172). A total of 18 MG102-71- 93 -#137172Attorney Docket No : 00010.037.1801 ABE variants were compared to ABE-192 (amino acid sequence denoted by SEQ ID NO: 289) and as shown in FIG. 7A, 17 variants outperformed at AAVS 1. Based on the combined activities observed, the study identified the MG102-71 N599 position as an improved inlay site for the ABE design.

[0244] Based on the fraction of mCherry -positive cells in the dually-transfected sub-population, the study identified improved MG34-29 ABE variants (SEQ ID NOs: 119-152 and 173-179). A total of 32 new MG34-29 ABE variants were compared to ABE-87 (contains the MG68-4 (D109N, T112R, A155R) deaminase inlaid within the MG34-29 nickase); and as shown in FIG. 7C, 5 variants outperformed in activity' at AAVS1. Based on the combined activity7, the study identified El 12 and L516 as improved inlay sites.

[0245] Combining the variants from SMART BE screening and improved adenine deaminase variants resulted in systems with up to 40% editing based on stop-to-sense codon conversion analysis performed using a mammalian fluorescence reporter encoding a therapeutic target.

[0246] HEK293T cell fluorescence screen results for MG 102-71 arginine scanning

[0247] Based on the fraction of mCherry-positive cells in the dually-transfected sub-population, the study identified improved MG102-71 ABE arginine point mutants (SEQ ID NOs. 10-32). A total of 23 new MG102-71 ABE arginine point mutants were compared to ABE-192 (SEQ ID NO: 289); and as shown in FIG. 7B, 20 arginine point mutants outperformed ABE- 192. Inclusion of the arginine point mutations provided a further 1.4-fold activity boost, enabling the SMART BE to achieve up to 73% apparent editing based on the top-to-sense codon conversion analysis.Example 4 - Small ABEs are active at AAVS1 in SkM cells

[0248] This Example describes AAVS1 targeting by small ABEs in SkM cells.

[0249] Human skeletal muscle cells (SkM) cell culture, transfections, and data analysis

[0250] SkM cells (ATCC &PCS-950-010) were cultured in Mesenchymal Stem Cell Basal Medium supplemented with Primary Skeleton Cell Muscle Grow th Kit for 1-2 passages prior to lipofection. SkM cells were transfected w ith ABE mRNA plus guide RNA hD_ABE_Ex45-gl2 (SEQ ID NOs. 180-181) at 1 :5 mRNA:gRNA molar ratio at four different mRNA masses. SkM cells were trypsinized and counted, and the equivalent volume to 12,500 viable cells were added to each well in a 96-well plate. Additional pre-equilibrated media was added to each well to bring the total volume to 100 pL. On the day of transfection, 1.5 pL of a transfection reagent, such as Lipofectamine Messenger Max Solution per pg of mRNA w as combined with OptiMEM media to make a total 50 pL master mix solution, vortexed. and incubated for at least 5 minutes at room temperature. In separate tubes, different masses of ABE-163 mRNA (400 ng, 200 ng, 100 ng and 50 ng) were mixed with 1 :5 molar ratio of hD_ABE_Ex45-gl2 guide RNA and combined with- 94 -#137172Attorney Docket No : 00010.037.1801 OptiMEM media to make a total 50 pL reaction volume. After a brief vortex, the appropriate volume of MessengerMax solution was RNA solution mix, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were incubated for 10 minutes at room temperature and added directly to the SkM cells. Three days post-transfection, the media was aspirated and genomic DNA was harvested and the resulting genomic DNA was used as a template for the NGS PCR. Targeted sequences were amplified with NGS primers. Amplicons around 250 bp long were checked, sequenced, and analyzed to measure gene editing outcomes.

[0251] Experimental Results

[0252] Rational engineering of the nickase chassis for SMART ABEs identified several improved variants active at the AAVS1 target locus. A dose titration experiment of SMART ABE variant mRNA indicated successful editing, with an A to G substitution rate of up to 13.44% at the AAV S 1 site with ABE- 158 (SEQ ID NO: 255) (FIG. 8) Further experiments with 400 ng of ABE mRNA for selected rationally engineered variants tested at AAVS1 in the SkM cells showed a marked increase in the base editing with MG34 family ABEs (ABE-211 (SEQ ID NO: 304), ABE-218 (SEQ ID NO: 311), ABE-267 (SEQ ID NO: 352), ABE-268 (SEQ ID NO: 353), ABE-269 (SEQ ID NO: 354), ABE-270 (SEQ ID NO: 355), ABE-271 (SEQ ID NO: 356), ABE-272 (SEQ ID NO: 357), and ABE-273 (SEQ ID NO: 358)) with a maximum editing of 60% at AAVS1 (FIG. 9A). For the MG102 family ABEs (ABE-245 (SEQ ID NO: 338), ABE-254 (SEQ ID NO: 347), ABE- 274 (SEQ ID NO: 204), and ABE-275 (SEQ ID NO: 210)), the study observed an A to G conversion of over 35% at AAVS1 (FIG. 9B).EQUIVALENTS

[0253] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.- 95 -#137172

Claims

Attorney Docket No : 00010.037.1801CLAIMSWHAT IS CLAIMED IS:

1. An engineered base editing system, comprising:(a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 210, 356, 182-209, 21 1-355, 357, and 358; and(b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence.

2. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 210, 356, 182-209, 211-355, 357, and 358.

3. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 210, 356, 182-209, 211- 355, 357, and 358.

4. The engineered base editing system of any one of claims 1-3, wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 31, 177, 2-8, 10-30, 32-176, 178, and 179.

5. The engineered base editing system of claim 4, wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 31, 177, 2-8, 10- 30, 32-176, 178, and 179.

6. The engineered base editing system of claim 4, wherein the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 31, 177, 2-8, 10-30, 32- 176, 178, and 179.

7. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 210, 272, 281, 289, 350, and 351.

8. The engineered base editing system of claim 7, wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 210, 272, 281, 289, 350, and 351.

9. The engineered base editing system of claim 7, wherein the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 210, 272, 281, 289, 350, and 351.- 96 -#137172Attorney Docket No : 00010.037.180110. The engineered base editing system of any one of claims 7-9, wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 31, 93, 102, 1 10, 171, and 172.

11. The engineered base editing system of any one of claims 7-9, wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 31, 93, 102, 110. 171, and 172.

12. The engineered base editing system of any one of claims 7-9, wherein the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 31, 93, 102, 110, 171, and 172.

13. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%. 99% or 100% identity to any one of SEQ ID NOs: 212, 213, 216, 229, 242, 255, 268, 276, 277, 284, 285, 293, 331 , and 356.

14. The engineered base editing system of claim 13, wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 212, 213, 216. 229, 242, 255. 268, 276, 277, 284, 285. 293, 331, and 356.

15. The engineered base editing system of claim 13, wherein the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 212, 213, 216, 229, 242, 255, 268, 276, 277, 284, 285, 293, 331, and 356.

16. The engineered base editing system of any one of claims 13- 15, wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 33, 34, 37, 50, 63, 76, 89, 97, 98, 105, 106, 114, 152, and 177.

17. The engineered base editing system of any one of claims 13-15. wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs:

33. 34, 37, 50, 63, 76, 89, 97, 98, 105, 106, 114, 152, and 177.

18. The engineered base editing system of any one of claims 13-15, wherein the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 33, 34, 37, 50, 63, 76, 89, 97, 98, 105, 106, 114, 152, and 177.

19. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-188.

20. The engineered base editing system of claim 19. wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 182-188.21 . The engineered base editing system of claim 19, wherein the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 182-188.- 97 -#137172Attorney Docket No : 00010.037.180122. The engineered base editing system of any one of claims 19-21. wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 2-8.

23. The engineered base editing system of any one of claims 19-21, wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity' to any one of SEQ ID NOs: 2-8.

24. The engineered base editing system of any one of claims 19-21. wherein the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 2-8.

25. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity' to any one of SEQ ID NOs: 189-211.

26. The engineered base editing system of claim 25. wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 189-21 1 .

27. The engineered base editing system of claim 25, wherein the base editor comprises a sequence having 100% sequence identity' to any one of SEQ ID NOs: 189-211.

28. The engineered base editing system of any one of claims 25-27. wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity7to any one of SEQ ID NOs: 10-32.

29. The engineered base editing system of any one of claims 25-27, wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity’ to any one of SEQ ID NOs: 10- 32.

30. The engineered base editing system of any one of claims 25-27, wherein the base editor is encoded by a nucleic acid sequence having 100% identity' to any one of SEQ ID NOs: 10-32.

31. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 212-297.

32. The engineered base editing system of claim 31, wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 212-297.

33. The engineered base editing system of claim 31. wherein the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 212-297.

34. The engineered base editing system of any one of claims 31-33, wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 33-118.

35. The engineered base editing system of any one of claims 31-33. wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 33- 118.- 98 -#137172Attorney Docket No : 00010.037.180136. The engineered base editing system of any one of claims 31-33. wherein the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 33-118.

37. The engineered base editing system of claim 1, wherein the base editor comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 298-358.

38. The engineered base editing system of claim 37. wherein the base editor comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 298-358.

39. The engineered base editing system of claim 37, wherein the base editor comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 298-358.

40. The engineered base editing system of any one of claims 37-39. wherein the base editor is encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 119-179.

41. The engineered base editing system of any one of claims 37-39, wherein the base editor is encoded by a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOs: 119- 179.

42. The engineered base editing system of any one of claims 37-39, wherein the base editor is encoded by a nucleic acid sequence having 100% identity to any one of SEQ ID NOs: 119-179.

43. The engineered base editing system of any one of claims 1-42, wherein the base editor comprises a deaminase.

44. The engineered base editing system of claim 43. wherein the deaminase binds non- covalently to the endonuclease.

45. The engineered base editing system of claim 43, wherein the deaminase is covalently linked to the endonuclease.

46. The engineered base editing system of claim 43. wherein the deaminase is fused to the endonuclease.

47. The engineered base editing system of any one of claims 1-46, wherein the engineered guide polynucleotide is a single guide nucleic acid.

48. The engineered base editing system of any one of claims 1-46, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

49. The engineered base editing system of any one of claims 1-48, wherein the engineered guide polynucleotide is RNA.

50. The engineered base editing system of any one of claims 1-49, wherein the endonuclease binds non-covalently to the engineered guide polynucleotide.51 . The engineered base editing system of any one of claims 1 -49, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.- 99 -#137172Attomey Docket No : 00010.037.180152. The engineered base editing system of any one of claims 1-51, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 180 or 181.

53. The engineered base editing system of any one of claims 1-52, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity' to SEQ ID NO: 180 or 181.

54. The engineered base editing system of any one of claims 1-53, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to SEQ ID NO: 180 or 181.

55. The engineered base editing system of any one of claims 1-54, wherein the base editor comprises a nickase domain.

56. The engineered base editing system of claim 55, wherein the nickase comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 362, residue 13 relative to SEQ ID NOs: 363, 364, or 366, residue 12 relative to SEQ ID NO: 365, residue 17 relative to SEQ ID NO:

367. residue 23 relative to SEQ ID NO: 368, or residue 10 relative to SEQ ID NO:

369. or any combination thereof.

57. The engineered base editing system of any one of claims 1-56, wherein the base editor further comprises a uracil DNA glycosylase inhibitor sequence.

58. The engineered base editing system of any one of claims 1-57, wherein the base editor further comprises a FAM72A sequence.

59. The engineered base editing system of claim 58, wherein the FAM72A sequence has at least 80% identity to SEQ ID NO: 370.

60. A nucleic acid encoding the engineered base editing system of any one of claims 1-59.

61. A vector comprising the nucleic acid of claim 60.

62. The vector of claim 61, wherein the vector is a plasmid, a minicircle, a CELiD, an adeno- associated virus (AAV) derived virion, a lentivirus, or an adenovirus.

63. A cell comprising the engineered base editing system of any one of claims 1-59, the nucleic acid of claim 60. or the vector of any one of claims 61-62.

64. The cell of claim 63, wherein the cell is a eukaryotic cell, a mammalian cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a prokaryotic cell.

65. The cell of claim 63, wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, SIP, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38. HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.

66. The cell of claim 63, wherein the cell is an engineered cell or a stable cell.- 100 -#137172Attorney Docket No : 00010.037.180167. A method for modifying a target nucleic acid sequence, comprising: contacting the target nucleic acid sequence using the engineered base editing system of any one of claims 1-59.

68. The method of claim 67, wherein modifying the target nucleic acid sequence comprises converting an adenine to a guanine in the target nucleic acid sequence.

69. The method of claim 67, wherein modifying the target nucleic acid sequence comprises converting a cytosine to a uracil in the target nucleic acid sequence.

70. The method of any one of claims 67-68, wherein the target nucleic acid sequence comprises deoxyribonucleic acid (DNA).

71. The method of any one of claims 67-70, wherein the target nucleic acid sequence comprises ribonucleic acid (RNA).

72. The method of any one of claims 67-71. wherein the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

73. The method of any one of claims 67-72, wherein the target nucleic acid sequence is modified in vitro, in vivo, or ex vivo.

74. The method of any one of claims 67-73, wherein the target nucleic acid sequence is modified within a cell.

75. The method of claim 74, wherein the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, a human cell, or a primary cell.

76. A method of modifying a nucleic acid encoding AAVS1 comprising contacting the nucleic acid sequence encoding AAVS1 with an engineered base editing system, said base editing system comprising: a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 182-358; and b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence.

77. The method of claim 76, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 180 or SEQ ID NO: 181.

78. Use of the engineered base editing system of any one of claims 1-59 for modifying a target nucleic acid sequence.- 101 -#137172

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