Compositions and methods for production and use of programmable base editors

Abstract

The present disclosure provides programmable base editors, and methods for their production and use. A subject programmable base editor includes (1) a CRISPR-Cas fusion protein comprising a CRISPR-Cas effector protein fused to a first member of a split-protein binding pair, where the CRISPR-Cas effector protein is a nickase (e.g., nCas9) or is catalytically inactive (e.g., dCas9, dCas12 such as dCas12a), and (2) a deaminase fusion protein comprising a deaminase protein fused to a second member of the split-protein binding pair. The CRISPR-Cas effector protein and the deaminase protein are not translationally fused to one another, but are instead post-translationally bound, e.g., in some cases covalently linked via a covalent bond (e.g., an isopeptide bond) formed between the two members of the split-protein binding pair (e.g. between a catcher protein and a partner tag protein).

Description

COMPOSITIONS AND ETHODS FOR PRODUCTION AND USE OF PROGRAMMABLE BASE EDITORSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 717,694 filed November 7, 2024, which application is incorporated herein by reference in its entirety.STATEMENT REGARDING RESEARCH FUNDING

[0002] Research for this invention was supported by awards from the Cystic Fibrosis Foundation.INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 546PR\ / _SeqList_v2.xml” created on November 6, 2024 and having a size of 222,181 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.I. INTRODUCTION

[0004] Programmable base editors are a class of genome editing effector proteins that can make precise, targeted changes to DNA base pairs in a narrow (~10 nt) window of genomic sequence without reliance on double-stranded breaks in chromosomal DNA. Base editor effectors can be delivered in several cargo formats: (i) DNA encoding the base editor protein as well as the guide RNA (typically delivered as plasmid or in a viral vector); (ii) mRNA encoding the base editor protein, co-delivered with guide RNA (typically delivered via lipid nanoparticle or via electroporation); (iii) as a pre-formed RNP enzyme comprising protein and guide RNA (typically delivered via electroporation). Base editor proteins include a deaminase fused to a CRISPR-Cas effector protein (e.g., nCas9). Base editor proteins are challenging to produce in high yields via recombinant expression in E. coli. This has limited its clinical use to mRNA / gRNA delivery; this is in stark contrast to Cas9 nuclease, which has been used in multiple clinical trials in its protein-based RNP format. An analogous example relates to the commercial availability of genome editing proteins: base editors cannot be purchased in protein format for research use, while Cas9 nuclease is sold by multiple vendors, even as a GMP-grade product suitable for clinical use.

[0005] It is unclear why high-yield production of base editor proteins has been an insurmountable challenge to date. There is a need for base editor proteins that are highly active and can be produced with high yield. Such is provided by the compositions and methods described herein.II. SUMMARY

[0006] The work described in the experimental examples below led to the surprising finding by the inventors that limitations associated with producing a CRISPR-Cas base editor can be overcome by preparing a CRISPR-Cas fusion protein and a deaminase fusion protein separately, and then placing them in contact with one another (i.e., contacting the CRISPR-Cas fusion protein with the deaminase fusion protein). Thus, provided here is a modular approach (and the associated components) for assembling high- yield, high-purity base editors (BEs) (e.g., adenine and cytosine BEs) via separated translation events followed by conjugation of the two constituent domains: a CRISPR- Cas effector protein (e.g., Cas9) (provided by a CRISPR-Cas fusion protein) and a deaminase (provided by a deaminase fusion protein). This approach is sometimes referred to herein as “BEaST” (Base Editor assembled via Separated Translation). A subject CRISPR-Cas fusion protein includes a CRISPR-Cas effector protein fused to a first member of a split-protein binding pair, where the CRISPR-Cas effector protein is a nickase (e.g., nCas9) or is catalytically inactive (e.g., dCas9, dCas12 such as dCas12a); while the deaminase fusion protein includes a deaminase protein fused to a second member of the split-protein binding pair. In other words, a subject CRISPR- Cas fusion protein includes a first member of a split-protein binding pair fused to a nickase CRISPR-Cas effector protein (e.g., nCas9), or fused to a dead CRISPR-Cas effector protein (e.g., dCas9, dCas12 such as dCas12a), and the deaminase fusion protein includes a second member of the split-protein binding pair fused to a deaminase protein. In some cases, the split-protein binding pair comprises a catcher / tag system (e.g., SpyCatcher / SpyTag). In some cases, the split-protein binding pair comprises a GFP1-10 / GFP11 system. The two members of the splitprotein binding pair spontaneously bind to one another, and thus upon contact with one another, the two separate fusion proteins bind to one another, forming a CRISPR- Cas base editor.

[0007] As an example, in some cases, when the split-protein binding pair comprises a catcher / tag system, the CRISPR-Cas effector protein (e.g., nCas9) is fused to (a) a catcher protein (e.g., a spy catcher protein), or (b) a partner tag protein (e.g., a spytag), while the deaminase protein is fused to the other of (a) and (b). For example, if the CRISPR-Cas effector protein is fused to the catcher protein (e.g., a spy catcher protein), then the deaminase protein (e.g., TadA-8e) is fused to the partner tag protein (e.g., a SpyTag), and vice versa. Upon contact with one another, the two separate fusion proteins become covalently linked via an isopeptide bond formed between the catcher protein and the partner tag protein. Thus, a CRISPR-Cas effector protein (e.g., nCas9) and a deaminase protein (e.g., TadA-8e) can be fused to a catcher / tag system such as SpyCatcher / SpyTag. The fusion proteins are independently prepared (and in some cases purified), and the fusion proteins are then brought into contact with one another, which triggers conjugation via isopeptide bond formation between the catcher / tag portions of the fusion proteins.

[0008] Provided are programmable base editors. Also provided are methods for producing a subject programmable base editor, and methods of using a subject programmable base editor to modify target DNA. Reagents, compositions, and kits / systems that find use in practicing the subject methods are provided.

[0009] The compositions and method disclosed herein have broad applications in gene editing and genetic research. The capacity for Cas9 nuclease protein to be produced at high yields allowed Cas9 enzyme (used as a protein / RNA complex, aka RNP) to be used in the clinic just 7 years after the first proof-of-concept report of the enzyme’s use in cells. This has been extremely effective: Cas9 RNP is responsible for dozens of patients being cured of hemoglobinopathies (one of CRISPR’s greatest clinical success stories). In contrast, base editors have not been widely used as RNP, almost certainly due to the extreme difficulty and unpredictability of base editor protein production. Research and biopharma efforts instead often rely on mRNA for base editor delivery, a cumbersome solution that is nevertheless more appealing than trying to make high yield base editor protein. The methods and compositions provided herein improve access to base editor protein, facilitating base editing efforts to use RNP, a more appealing cargo class than mRNA (which is technically challenging and can have negative impacts on cells).

[0010] The inventors realized that a challenge with making an intact base editor protein, e.g., in cells such as E. coli, is that the two fusion proteins have distinct optimal conditions for expression: CRISPR-Cas proteins such as Cas9 can call for “slow” expression (e.g., in some cases 12-16 h at ~16°C) while deaminase proteins (e.g., TadA such as TadA-8e) call for “quick” expression (e.g., in some cases ~4 h at 37°C). The inventors’ solution sidesteps this problem. This approach (conjugation following independentexpression & purification) facilitates high-yield production of highly enriched (e.g., extremely pure), active (e.g., fully active) base editor proteins. This approach allows the user to control the starting reagents and protocols for conducting the conjugation of the fusion proteins. In some cases, the two fusion proteins are produced in sufficient quantities to conduct reaction optimization trials. In some cases, one or more subsequent purification steps are performed to remove unwanted byproducts of the reaction, e.g., with the goal of removing as much un-coupled protein as possible. The compositions and methods disclosed herein can be scalable and suitable for industrial production.III. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0012] FIG. 1 SpyTag / Spycatcher and site-specific TadA-Cas9 coupling. Schematic of an example embodiment using conjugation following independent expression & purification (CFIEP) to generate a chimeric Cas9 protein (a base editor in this example embodiment) comprising a deaminase (TadA deaminase) and a Cas9 nickase. In this particular example, spontaneous isopeptide bond formation occurs in Spy CnaB2 domain to produce SpyTag and SpyCatcher affinity tags recombinantly introduced to a TadA deaminase and a Cas9 nickase, together generating a base editor.

[0013] FIG. 2 SpyTag / Spycatcher was conjugated TadA and nCas9 to form a base editor. Lanes 1 and 2: constituent domains of the base editor on SDS-PAGE; each domain bears SpyTag or SpyCatcher, making each compatible with SpyTag / SpyCatcher conjugation (no high-affinity SpyTag / Spycatcher tags added; no conjugation). Lanes 3 and 4: after mixing base editor domains together (Cas9 nickase and TadA deaminase), band corresponding to base editor MW appeared, demonstrating proof- of-concept conjugation (3); subsequent purification steps removed unwanted byproducts to achieve high purify base editor (4).

[0014] FIG. 3 Percent editing efficiencies of CFIEP-made and commercial ABE B2M- knockout via PERC in primary human T cells, (left) Adenine base editor (ABE) B2M- RNPs engineered by conjugation following independent expression and purification (CFIEP), compared to a commercially available ABE supplied by a contract researchorganization (CRO). The ABEs were delivered via peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC; see, e.g., Foss et al., Nat Biomed Eng. 2023;7(5):647-660). Knockout efficiencies were assayed 4 days after delivery by flow cytometry. 50 pmol of RNP were delivered, (middle) ABE edited cell yield measured by total number of B2M-knockout cells, (right) Primary human T cells were stained with live-dead stain and surface marker-targeting antibodies and sampled at defined volumes to quantify cell counts; NT = no treatment.

[0015] FIG. 4 depicts amino acid sequences of various constructs generated by the inventors.

[0016] FIG. 5. Comparison of protein yields from recombinantly expressed adenine base editor. Adenine base editors (ABEs) were expressed and purified by four different sources. Commercially available ABE was supplied by a contract research organization (CRO). The QB3 MacroLab and Wilson Lab (WL) also prepared ABEs by single-plasmid expression and 3-step purification in contrast to ABEs engineered by conjugation following independent expression and purification (CFIEP); n=3.

[0017] FIG. 6. Relative editing efficiencies mediated by commercially- or CFIEP-produced cytosine base editor in primary human T cells. Primary human T cells were edited using 100 pmol of cytosine base editor (CBE) RNP targeting the T cell receptor beta chain (TRBC) delivered via A5K peptide (10 uM). Three days after editing, cells were stained and analyzed using flow cytometry for T cell receptor (TCR) knockout. Comparison of a commercially purchased CBE shows comparable knockout rates with the CBE engineered by conjugation following independent expression and purification (CFIEP).

[0018] FIG. 7A-7D. The BEaST strategy for base editor production, (a) A diagram representing an adenine base editor (ABE) generated via the BEaST strategy. The white (unfilled) shape represents an inactive TadA (deaminase) domain, while the similar purple (filled) shape represents an active TadA domain, (b) Comparison of ABE enzymes purified as genetic fusion (traditional ABE or tABE) or via the BEaST strategy, revealing improved purity of BEaST protein, (c) Protein yield expressed as mg of pure ABE protein per liter of E. coli culture. Lower-left panel: tABE production, wherein CRISPR nickase domain is fused to TadA deaminase domain(s) for recombinant expression in E. coli, followed by purification. Lower-right panel: ABE production via the BEaST strategy, wherein the CRISPR nickase domain and TadA deaminase domain(s) are produced via separate translation events, resulting in two polypeptides capable of assembling via (in one example) interaction between theirrespective fused SpyCatcher and SpyTag polypeptide domains. This figure represents one example of how to use the BEaST strategy; importantly, both plasmids could be co-transformed into a single E. coli cell population, or both gene cassettes can be incorporated into a single plasmid that can be transformed into a single E. coli population. In such examples, assembly of the two domains may happen within E. coli cells. In the example represented here, assembly of the two domains occurs following partial purification of each domain from distinct E. coli cell populations.

[0019] FIG. 8A-8E. ABE produced via the BEaST strategy exhibits activity similar to - or better than - that of tABE. (a) Diagram representing the experimental design: ABE RNP (targeting the B2M gene to alter splicing and induce knockout) is delivered to primary human T cells via either electroporation or peptide-enabled RNP-format CRISPR (PERC) and editing outcomes are assessed via next-generation DNA sequencing or flow cytometry. Matched amounts of RNP was delivered in each experiment. The gRNA targets sequence CTTACCCCACTTAACTATCT (SEQ ID NO: 169). (b) In a pilot study, the BEaST ABE exhibited editing efficiency surpassing that of tABE produced by a contract research organization (CRO). (c) Diagrams representing the distinct enzymes tested. Empty (white) or filled (purple) shapes communicate the number of active domains: either 1 , 2, or 4 per ABE RNP. “NG” denotes an ABE incorporating a Cas9 nickase evolved to recognize an NG PAM. The cBEaST enzyme (cytidine base editor produced via the BEaST strategy) incorporates a TadA domain evolved to deaminate cytidine in the context of DNA (as opposed to RNA). “QT” refers to an evolved deaminase domain (sometimes referred to as ABE9) that is based on the “8e” version of TadA and incorporates two additional amino acid point mutations: N108Q & L145T. “TTI” refers to an evolved deaminase domain that is based on the “8e” version of TadA and incorporates three amino acid point mutations: S7T, A114T, F148I. (d) Activity of ABE enzymes delivered into primary human T cells in RNP format via electroporation. BEaST ABE enzymes perform comparably to tABE enzymes. This also demonstrates robust activity of the cBEaST enzyme in RNP format, (e) Activity of ABE enzymes delivered into primary human T cells in RNP format via PERC. BEaST ABE enzymes perform comparably to tABE enzymes, and sometimes exhibit improved activity. This also demonstrates robust activity of the cBEaST enzyme in RNP format.

[0020] FIG. 9A-9C. Genome editing in primary human T cells via BEaST ABE or tABE RNPs. RNPs were delivered via electroporation, and the gRNA targeted the erythroid- specific enhancer of BCL11a. (b) The two gRNA target sequences employed, withdeamination candidate adenines highlighted. Each gRNA is named for the NGG PAM it relies on. (c) Editing results as assessed by next-generation DNA sequencing. Each bar represents the editing rate at a specific adenine nucleotide. Top row: editing results using the “AGG” gRNA. Bottom row: editing results using the “GGG” gRNA. Left panels: absolute rates of A-to-G conversion. Right panels: normalized rates of A- to-G conversion, intended to represent the distribution of edits regardless of the absolute editing efficiency. These results demonstrate an example of BEaST RNPs performing with high efficiency (sometimes surpassing tABE). Furthermore, these results allow comparison of the efficiency across the base editing “window” for each enzyme. This reveals that BEaST ABE enzymes are comparable - but not identical - to tABE in their activity.

[0021] FIG. 10A-10E. BEaST ABE mediates efficient genome editing of brain neurons in vivo, (a) The reporter cassette incorporated in the GER 10 mouse model: adenine base editing can remove a premature stop codon, allowing translation of the Venus GFP gene, (b) High efficiency editing following administration of ABE RNP produced using the BEaST strategy. BEaST ABE was administered into the right hemisphere via intrastriatal / intraparenchymal convection-enhanced delivery (CED). The left hemisphere received a similar administration of buffer, (c) Quantification of the area with GFP expression (resulting from BEaST ABE activity); on average, over 15% of the striatal area was edited based on three analyzed sections, (d) Quantification of neurons that express GFP (resulting from BEaST ABE activity); on average, over 35% of neurons were edited based on three analyzed sections, (e) Histology demonstrating robust GFP expression following BEaST ABE administration. DAPI was used to identify nuclei; NeuroTrace was used to identify neurons.

[0022] FIG. 11. The BEaST system is functional when two distinct plasmids are used for expression of components in the same cell - in this case a single population of E. coli cells. This SDS-PAGE gel (coomassie stained) shows partial purification of BEaST ABE expressed in a single population of E. coli cells following transformation using two plasmids: one encoding a tandem deaminase gene with a C-terminal SpyTag, the other encoding a Cas9 nickase gene with an N-terminal SpyCatcher tag. Using a nickel affinity column, the histidine-tagged BEaST ABE protein was eluted (lane 4), demonstrating that the BEaST polypeptides can assemble within the E. coli cell (or in cell lysate) following expression using two plasmids in the same cell population.

[0023] FIG 12. Plasmid map diagram representing the encoded BEaST system components on a single DNA: in this example embodiment, two tandem ORFs encoding (i) tandemdeaminase gene (labeled as “TadA dimer”) with a C-terminal SpyTag, followed by (ii) a Cas9 nickase gene with an N-terminal SpyCatcher tag. Each gene has its own T7 promoter and lac operator with one transcription terminator present at the 3’ end of the Cas9 gene. This plasmid construct allows for two separate transcripts to be produced. The first transcript includes both TadA dimer and Cas9 (resulting in two polypeptides following translation); the second transcript only includes Cas9 (one polypeptide following translation).

[0024] FIG 13. The BEaST system is functional when a single DNA (e.g., plasmid) is used for expression of BEaST components in a single population of E. coli cells. This SDS- PAGE gel (coomassie stained) shows evidence of assembled BEaST ABE resulting from component expression in a single population of E. coli cells following transformation using one plasmid encoding both (i) a tandem deaminase gene with a C-terminal SpyTag and (ii) a Cas9 nickase gene with an N-terminal SpyCatcher tag. This demonstrates that the BEaST polypeptides can assemble within the E. coli cell (or in cell lysate) following component expression using a single plasmid.IV. DEFINITIONS

[0025] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to all forms of nucleic acid (e.g., oligonucleotides) including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA, IncRNA, RNA antagomirs, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides also include non-coding RNA, which include for example, but are not limited to, RNAi, miRNAs, IncRNAs, RNA antagomirs, aptamers, and any other non-coding RNAs known to those of skill in the art. Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides as well as analogues and derivatives. The term "polynucleotide" also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof, and is synonymous with nucleic acid sequence. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment as described herein encompassing a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0026] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA], In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule: guanine (G) can also base pair with uracil (U). For example, G / U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a guanine (G) is considered complementary to both an uracil (U) and to an adenine (A). For example, when a G / U base-pair can be made at a given nucleotide position of a dsRNA duplex, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0027] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide cancomprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.

[0028] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0029] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0030] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0031] "Binding" as used herein (e.g. with reference to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA and a target nucleic acid; and the like). While in a state of non- covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequencespecific. Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10-6M, less than 10'7M, less than 10-8M, less than 10'9M, less than 10’10M, less than 10-11M, less than 10-12M, less than 10-13M, less than 10-14M, or less than 10-15M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0032] By "binding domain" it is meant a protein domain that is able to bind non-covalently to another molecule. A binding domain can bind to, for example, an RNA molecule (an RNA-binding domain) and / or a protein molecule (a protein-binding domain). In the case of a protein having a protein-binding domain, it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more regions of a different protein or proteins.

[0033] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic sidechains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagineglutamine. Coded amino acids (followed in parentheses by their corresponding three- letter codes and one-letter codes) include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F); proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), or valine (Vai; V).

[0034] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / , http: / / www.sbg.bio.ic.ac.uk / ~phyre2 / . See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0035] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and / or regulate translation of an encoded polypeptide.

[0036] As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive transcription.

[0037] The term "naturally-occurring" or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[0038] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps resulting in a construct having a structural coding or noncoding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", above). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant,etc ). Thus, a "recombinant" polypeptide is the result of human intervention, but may have a naturally occurring amino acid sequence.

[0039] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.

[0040] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression (the coding sequence can also be said to be operably linked to the promoter).

[0041] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0042] “Heterologous,” as used herein, refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, relative to a CRISPR-Cas effector protein of the present disclosure, a heterologous polypeptide comprises an amino acid sequence from a protein other than that CRISPR-Cas effector protein. As such, a CRISPR-Cas effector protein of the present disclosure can be fused to polypeptide (an amino acid sequence) from a non-CRISPR-Cas effector protein (e.g., an NLS, a member of a split-protein binding pair, a linker, a protein tag such as a His tag or flag tag, and the like), and the sequence of the fused polypeptide could be considered a heterologous polypeptide (it is heterologous to the CRISPR- Cas effector protein). As another example, a guide sequence of a guide RNA that is heterologous to a protein-binding sequence (a constant region / scaffold) of a guide RNA is a guide sequence that is not found in nature together with that protein-binding sequence.

[0043] As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs, or that in which it was produced. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and / or in which the compound of interest is partially or substantially purified. For example, an"isolated" protein, plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components present where the substance or a similar substance naturally occurs or from which it is initially prepared. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated.

[0044] An isolated protein, plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure. As used herein, the term “purified” or “substantially purified” refers to a compound that is removed from its natural environment (or from the environment in which it was produced) and is enriched - is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated, or associated with as a result of its production. As an example, a protein of interest (e.g., a heterologous protein) can be produced in bacterial cells, and the cells can be lysed in order to extract, isolate, and purify (enrich for) the protein of interest.

[0045] The term “isopeptide bond” is used herein to mean a covalent amide bond in a protein connecting a side chain to a side chain, or connecting a side chain to a protein's main chain. As an example, an isopeptide bond is formed when a catcher protein (e.g., a SpyCatcher such as SpyCatcher3) forms a spontaneous bond upon binding its partner tag protein (e.g., a SpyTag such as SpyTag3). See, e.g., FIG. 1.

[0046] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0047] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0048] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0049] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0051] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0052] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. As such, the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0053] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0054] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.V. DETAILED DESCRIPTION

[0055] The present disclosure provides programmable base editors, methods for producing a subject programmable base editor, and methods of using a subject programmable base editor to modify target DNA. A subject programmable base editor includes (1) a CRISPR-Cas fusion protein comprising a CRISPR-Cas effector protein fused to a first member of a split-protein binding pair, where the CRISPR-Cas effector protein is a nickase (e.g., nCas9) or is catalytically inactive (e.g., dCas9, dCas12 such as dCas12a); and (2) a deaminase fusion protein comprising a deaminase protein fused to a second member of the split-protein binding pair. The CRISPR-Cas effector protein and the deaminase protein are not translationally fused to one another, but are instead post-translationally bound, e.g., in some cases covalently linked via a covalent bond (e.g., an isopeptide bond) formed between the two members of the split-protein binding pair (e.g. between a catcher protein and a partner tag protein). Thus, as noted above, provided here is a modular approach (and the associated components) for assembling high-yield, high-purity base editors (BEs) (e.g., adenine and cytosine BEs) via separated translation events followed by conjugation of the two constituent domains: a CRISPR-Cas effector protein (e.g., Cas9) (provided by a CRISPR-Cas fusion protein) and a deaminase (provided by a deaminase fusion protein). This approach is sometimes referred to herein as “BEaST” (Base Editor assembled via Separated Translation).

[0056] The compositions and methods herein facilitate retention of full activity of the deaminase protein (i.e. , the deaminase retains full activity once assembled as part of a programmable base editor). For example, in some cases a subject programmable base editor has 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) deaminase activity compared to the deaminase activity of the deaminase fusion protein prior to conjugation with the CRISPR-Cas fusion protein. In other words, in some cases, the programmable base editor that is produced retains 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) deaminase activity when compared to the deaminase activity of the deaminase fusion protein prior to contact with the CRISPR-Cas fusion protein. In some cases, a subject programmable base editor has 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) deaminase activity compared to the deaminase activity of the deaminase fusion protein prior toconjugation with the CRISPR-Cas fusion protein. In some cases, a subject programmable base editor has 98% or more (e.g., 99% or more, or 100%) deaminase activity compared to the deaminase activity of the deaminase fusion protein prior to conjugation with the CRISPR-Cas fusion protein.Split-protein binding pairs

[0057] As noted above, each fusion protein (CRISPR-Cas fusion protein and deaminase fusion protein) includes one member of a split-protein binding pair, i.e., one member of the split-protein binding pair is fused (e.g., translationally fused) to the CRISPR-Cas effector protein (nickase, e.g., nCas9, or catalytically inactive, e.g., dCas9, dCas12 such as dCas12a), and the other member of the split-protein binding pair is fused (e.g., translationally fused) to the deaminase protein. The two members of the splitprotein binding pair spontaneously bind to one another, and thus upon contact with one another the two separate fusion proteins (the CRISPR-Cas fusion protein and the deaminase fusion protein) bind to one another, forming a CRISPR-Cas base editor.

[0058] In some cases, the first member of the split-protein binding pair (e.g., a SpyCatcher protein) is fused N-terminal to the CRISPR-Cas effector protein (e.g., nCas9). In some cases, the first member of the split-protein binding pair (e.g., a SpyCatcher protein) is fused C-terminal to the CRISPR-Cas effector protein. In some cases, the second member of the split-protein binding pair (e.g., a SpyTag protein) is fused N-terminal to the deaminase protein. In some cases, the second member of the split-protein binding pair (e.g., a SpyTag protein) is fused C-terminal to the deaminase protein. In some cases, the first member of the split-protein binding pair (e.g., a SpyCatcher protein) is fused N-terminal to the CRISPR-Cas effector protein; and the second member of the split-protein binding pair (e.g., a SpyTag protein) is fused C-terminal to the deaminase protein.Catcher / tag system

[0059] In some cases, the split-protein binding pair comprises a catcher / tag system (e.g., SpyCatcher / SpyTag). As would be understood to one of ordinary skill in the art, catcher proteins (e.g., a SpyCatcher such as SpyCatcher3) form a spontaneous amide bond upon binding their partner tag protein (e.g., a SpyTag such as SpyTag3) (see, e.g., FIG. 1). The catcher / tag sequences (e.g., SpyCatcher / SpyTag) are thereby covalently bonded by an isopeptide bond. As noted above, the term “isopeptide bond”is used herein to mean a covalent amide bond in a protein connecting a side chain to a side chain, or connecting a side chain to a protein's main chain.

[0060] Any convenient catcher / tag system (catcher protein / partner tag protein combination) can be used, and many such combinations are known in the art. Examples of catcher proteins include, but are not limited to: SpyCatcher [e.g., SpyCatcherOOl (also referred to as SpyCatcherl), SpyCatcher002 (also referred to as SpyCatcher2), SpyCatcher003 (also referred to as SpyCatcher3), SpyCatcher AN 1AC1 , SpyCatcher- Dodecin, SpyCatcher-mi3, and the like], SpyLigase, SnoopCatcher, SnoopLigase, and SpyDock. An example of a catcher protein is SpyCatcher3: VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT (SEQ ID NO: 11).Examples of partner tag proteins (that correspond to catcher proteins) include, but are not limited to: SpyTag [e.g., SpyTagOOl , also referred to as SpyTagl (AHIVMVDAYKPTK; SEQ ID NO: 1), SpyTag002, also referred to as SpyTag2 (VPTIVMVDAYKRYK; SEQ ID NO: 4), SpyTag003 (also referred to as SpyTag3) (RGVPHIVMVDAYKRYK) (SEQ ID NO: 7)], KTag (ATHIKFSKRD; SEQ ID NO: 2), SnoopTag (KLGDIEFIKVNK; SEQ ID NO: 3), SnoopTagJr (KLGSIEFIKVNK; SEQ ID NO: 5), and DogTag (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 6).

[0061] As used herein, the term “SpyCatcher” and “SpyTag” are meant to encompass any version of the SpyCatcher system series, e.g., version 1 (US 9,547,003), version 2 (WO 2018 / 197854)(SpyCatcher2, SpyTag2) and version 3 (WO 2020 / 183198)(SpyCatcher3, SpyTag3). If the original SpyCatcher or SpyTag are intended, the terms SpyCatcherl (or SpyCatcherOOl) or SpyTagl (or SpyTagOOl) will be used.

[0062] For more information related to catcher / tag systems (catcher proteins and their partner tag proteins), see, e.g., Hatlem et al., Int J Mol Sci. 2019 Apr 30;20(9):2129 [see, e.g., Table 1 of Hatlem et al.]; Li et al., J. Mol. Biol. 2014, 426, 309-317; Fierer et al., Proc. Natl. Acad. Sci. USA 2014, 111 , E1176-E1181; Veggiani et al., Proc. Natl. Acad. Sci. USA 2016, 113, 1202-1207; Keeble et al., Angew. Chem. Int. Ed. Engl. 2017, 56, 16521-16525; Buldun et al., J. Am. Chem. Soc. 2018, 140, 3008- 3018; Anuar et al., Nat. Commun. 2019, 10, 1743; Keeble et al., Proc Natl Acad Sci U S A. 2019 Dec 26;116(52):26523-26533; and Zakeri et al. Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7. Also see U.S. Patent Nos. 9547003, 11059867 and 12130292, and patent application publication Nos. US20240327819,US20200010513, US20200407751 , US20220002695, US20220010337, US20240209355, US20240101987, US20240327527, US20180244730, WO2018197854, W02017070742, WO2016193746, and WO2020183198; all of which are incorporated herein by reference for their disclosures related to catcher proteins and their partner tag proteins.GFP1-10 / GFP11 system

[0063] In some cases, the split-protein binding pair comprises a GFP1-10 / GFP11 system. When the first ten p-sheets of a fluorescent protein (referred to herein as “GFP1-10” or “GFP1.10”) and the 11th [3-sheet (referred to herein as “GFP11” or “GFPn”) are in contact, they will self-assemble and reconstitute a fluorescent protein (e.g., GFP). Without wishing to be bound by theory, the GFP1-10 fragment has the three residues that constitute the GFP chromophore, but is non-fluorescent by itself because chromophore maturation requires the conserved E222 residue located on the GFP11 fragment. See, e.g., Cabantous et al., Nat. Biotechnol. 23, 102-107 (2005).

[0064] As noted above, such a system is referred to herein as a GFP1-10 / GFP11 system. This term is intended to encompass any fluorescent GFP-like protein (not just “GFP”) that has this self-assembly property (see, e.g., Kamiyama, et al., Nat Commun. 2016; 7:11046). One member of this split-protein binding pair is referred to as “GFP1-10” while the other is referred to as “GFP11”.

[0065] Thus, in some cases a subject CRISPR-Cas fusion protein includes a GFP1-10, and the deaminase fusion protein includes a GFP11. The GFP1-10 is in some cases located N-terminal to the CRISPR-Cas effector protein (nickase, e.g., nCas9, or catalytically inactive, e.g., dCas9, dCas12 such as dCas12a). The GFP1-10 is in some cases located C-terminal to the CRISPR-Cas effector protein. The GFP11 is in some cases located N-terminal to the deaminase protein. The GFP11 is in some cases located C-terminal to the deaminase protein.

[0066] In some cases, a subject CRISPR-Cas fusion protein includes a GFP11 , and the deaminase fusion protein includes a GFP1-10. The GFP11 is in some cases located N-terminal to the CRISPR-Cas effector protein (e.g., nCas9). The GFP11 is in some cases located C-terminal to the CRISPR-Cas effector protein. The GFP1-10 is in some cases located N-terminal to the deaminase protein. The GFP1-10 is in some cases located C-terminal to the deaminase protein.

[0067] As an illustrative, but non-limiting example, in some cases the CRISPR-Cas fusion protein includes a GFP1-10 located N-terminal to the CRISPR-Cas effector protein(e.g., nCas9) (e.g., GFPi-io-nCas9) and the deaminase fusion protein includes a GFP11 located C-terminal to the deaminase protein (e.g., a TadA protein such as TadA-8e) (e.g., TadA-8e-GFPn).CRISPR-Cas effector proteins and guide RNAs

[0068] In class 2 CRISPR systems, the functions of the effector complex (e.g., the cleavage of target DNA) are carried out by a single protein (which can be referred to as a CRISPR-Cas effector protein) - where the natural protein is an endonuclease (e.g., see Zetsche et al, Cell. 2015 Oct 22;163(3):759-71 ; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385- 97; Shmakov et al., Nat Rev Microbiol. 2017 Mar;15(3):169-182: “Diversity and evolution of class 2 CRISPR-Cas systems”; Koonin et al., Curr Opin Microbiol. 2017 Jun:37:67-78; and Makarova et al., Nat Rev Microbiol. 2020 Feb;18(2):67-83;). As such, the term “class 2 CRISPR-Cas protein” or “CRISPR-Cas effector protein” is used herein to encompass the effector protein from class 2 CRISPR systems - for example, type II CRISPR-Cas proteins (e.g., Cas9), type V CRISPR-Cas proteins (e.g., Cpf1 / Cas12a, C2c1 / Cas12b, C2C3 / Cas12c, Cas12d / CasY, Cas12e / CasX), and type VI CRISPR-Cas proteins (e.g., C2c2 / Cas13a, C2C7 / Cas13c, C2c6 / Cas13b). Class 2 CRISPR-Cas effector proteins include type II, type V, and type VI CRISPR- Cas proteins, but the term is also meant to encompass any class 2 CRISPR-Cas protein suitable for binding to a corresponding guide RNA and forming a ribonucleoprotein (RNP) complex. In some cases the CRISPR-Case effector protein is a Cas13 protein and targets RNA.

[0069] A nucleic acid that binds to a class 2 CRISPR-Cas effector protein (e.g., a Cas9 protein; a type V or type VI CRISPR-Cas protein; a Cas12 protein; etc.) (thereby forming a ribonucleoprotein complex (RNP)) and targets the complex to a specific location within a target nucleic acid is referred to herein as a “guide RNA” or “CRISPR-Cas guide nucleic acid” or “CRISPR-Cas guide RNA.” A guide RNA can be used to guide the protein to the target sequence. It is to be understood that in some cases, a hybrid DNA / RNA can be made such that a guide RNA includes DNA bases in addition to RNA bases - but the term “guide RNA” is still used herein to encompass such hybrid molecules.

[0070] A nucleic acid molecule (e.g., a crRNA or a hybridized crRNA / tracrRNA) that binds to a CRISPR-Cas effector protein (e.g., a Cas9, a Cas12 such as Cas12a, a Cas13, etc.), forming a ribonucleoprotein complex (RNP), and targets the complex to aspecific target sequence within a target nucleic acid (e.g., target DNA or target RNA) is referred to herein as a “guide RNA” or “gRNA.” It is to be understood that in some cases, a hybrid DNA / RNA can be made such that a guide RNA includes DNA bases in addition to RNA bases - but the term “guide RNA” is still used herein to encompass such hybrid molecules. A guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a “guide sequence” (also referred to as a “targeting sequence” or a “spacer”), which is a nucleotide sequence that is complementary to (and hybridizes to) a target sequence of a target nucleic acid, e.g., a target DNA, (and thereby can be said to “target” a specific sequence or “target” a specific gene). The guide sequence can be changed each time a new target sequence is selected. A guide RNA also includes a portion that interacts with (binds to) the CRISPR-Cas effector protein. Because this region does not need to change each time a new target sequence is selected, this region is referred to as a “constant region” or “scaffold” (or “handle” or “repeat” or “protein-binding segment”). As would be known to one of ordinary skill in the art, depending on which type of CRISPR-Cas effector protein / system is being used, in some cases, the constant region is 5’ of the guide sequence (i.e., the guide sequence is 3’ of the constant region), and in other cases, the constant region is 3’ of the guide sequence (i.e., the guide sequence is 5’ of the constant region).

[0071] A guide RNA can be referred to by the protein to which it corresponds. For example, when a CRISPR-Cas effector protein is a Cas9 protein, the corresponding guide RNA can be referred to as a “Cas9 guide RNA.” Likewise, when a CRISPR-Cas effector protein is a Cas12a protein, the corresponding guide RNA can be referred to as a “Cas12a guide RNA,” and when a CRISPR-Cas effector protein is a Cas13 protein, the corresponding guide RNA can be referred to as a “Cas13 guide RNA.”

[0072] As will be known to one of ordinary skill in the art, in some embodiments, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” (or tracrRNA and crRNA) and can be referred to as a “dual guide RNA”, a “doublemolecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some embodiments, the guide RNA is one molecule (e.g., for some class 2 CRISPR-Cas proteins, the corresponding natural guide RNA is a single molecule; and in some cases, an activator and targeter (crRNA and tracrRNA) can be covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”

[0073] A gRNA can be selected using software. As a non-limiting example, considerations for selecting a gRNA can include, e.g., the PAM sequence for the CRISPR-Cas effector protein to be used, and strategies for minimizing off-target modifications. Tools, such as NUPACK and the CRISPR Design Tool, can provide sequences for preparing the gRNA, for assessing target modification efficiency, and / or assessing cleavage at off- target sites.

[0074] As would be understood to one of ordinary skill in the art, the guide RNA can be introduced into a cell as an RNA (or as a DNA / RNA hybrid), or can be introduced as a nucleic acid encoding the RNA (e.g., a DNA such as an expression vector such as a viral, plasmid, or minicircle DNA), in which case the cell transcribes the RNA from the introduced DNA. In some cases, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., a Pol III promoter such as U6 or H1). In some cases, one or more guide RNAs (e.g., 1 , 2, 3, 4, 5, 6, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2- 10, 2-8, 2-6, 2-5, 2-4, 3-10, 3-8, 3-6, 3-5, two or more, three or more, four or more, or five or more) (or nucleotide sequences that encode said guide RNAs) can be introduced into the same cell (e.g., to target different sequences of the same target nucleic, to target different target nucleic acids, etc.).

[0075] Scaffold sequences for various CRISPR-Cas guide RNAs are known in the art. For example, in some cases, the portion of the targeter-RNA (e.g., crRNA) that contributes to the scaffold (i.e. , is 3’ of the guide sequence) (e.g., when using an S. pyogenes Cas9 protein) includes: 5’-GUUUUAGAGCUAUGCUGUUUUG-3' (SEQ ID NO: 150). In some cases, it includes: 5’-GUUUUAGAGCUA-3' (SEQ ID NO: 151). in some cases, the activator-RNA (e.g., tracrRNA) (e.g., when using an S. pyogenes Cas9 protein) includes: ’5- AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG GCACCGAGUCGGUGCUU-3' (SEQ ID NO: 152). In some cases (e.g., when using an S. pyogenes Cas9 protein) a sgRNA includes 5’- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO: 153). In some cases (e.g., when using an S. pyogenes Cas9 protein) a sgRNA includes 5’- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAGUGGCACCGAGUCGGUGCUU-3' (SEQ ID NO: 154). Mutations / variants of the above sequences can also be used and many suitable examples will be known to one of ordinary skill in the art.

[0076] Examples of crRNA repeat sequences (also known as the scaffold) for Cas12a proteins include:LbCas12a crRNA:5’ AAUUUCUACUAAGUGUAGAU 3’ (SEQ ID NO: 155) - [spacer] 3’ AsCas12a crRNA:5’ AAUUUCUACUCUUGUAGAU 3’ (SEQ ID NO: 156) - [spacer] 3’ FnCas12a crRNA:5’ AAUUUCUACUGUUGUAGAU 3’ (SEQ ID NO: 157) - [spacer] 3’ PmCas12a crRNA:5’ AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 158) - [spacer] 3’ MbCas12a / Mb2Cas12a / Mb3Cas12a crRNA:5’ AAUUUCUACUGUUUGUAGAU 3’ (SEQ ID NO: 159) - [spacer] 3’ TsCas12a crRNA5’ AAUUUCUACUGUUGUAGAU 3’ (SEQ ID NO: 160) - [spacer] 3’ BsCas12a crRNA5’ AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 161) - [spacer] 3’

[0077] The following sequences are each an example of a scaffold of a naturally existing Cas13a guide RNA (e.g., a scaffold that is 5’ of the guide sequence) (See, e.g., Feng et al., Anal Chem. 2023 Jan 10;95(1):206-217):GUAAGAGACUACCUCUAUAUGAAAGAGGACUAAAAC (SEQ ID NO:162) (Listeria seeligeri) (“Lse”) (LseCas13a)GAUAUAGACCACCCCAAUAUCGAAGGGGACUAAAAC (SEQ ID NO: 163) (Leptotrichia shahii) (“Lsh”) (LshCas13a)AUUUAGACCACCCCAAAAAUGAAGGGGACUAAAAC (SEQ ID NO:164) (Leptotrichia buccalis) (“Lbu”) (LbuCas13a)GACCACCCCAAAAAUGAAGGGGACUAAAAC (SEQ ID NO: 165) (Leptotrichia buccalis) (“Lbu”) (LbuCas13a)GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC (SEQ ID NO:166) (LwaCas13a)GUCACAACUCCCAUGUAGGCGGAGACUGCAAC (SEQ ID NO: 167) (TccCas13a)GGAUUUAGAGUACCCCAAAAAUGAAGGGGACUAAAAC (SEQ ID NO:168) (LtrCas13a)

[0078] In the present disclosure, a subject CRISPR-Cas effector protein (e.g., a Cas9 protein, a Cas12 protein such as a Cas12a protein, a Cas13 protein, and the like) has reduced catalytic activity (e.g., a Cas9 protein with nickase activity (nCas9), a dead Cas9 protein (dCas9), a dead Cas12 protein (dCas12)). When the protein has nickase activity, it can be referred to as a nickase or an “nCas” (e.g., nCas9). When the protein binds to but does not cleave the target nucleic acid, it is referred to as “catalytic inactive”, which is used interchangeably with the term “dead”. A dead CRISPR-Cas effector protein can also be referred to as a “dCas” (e.g., dCas9, dCas12 such as dCas12a, dCas13, and the like).

[0079] For example, when a Cas9 protein has a mutation at one or more amino acid positions corresponding to D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or a A987 of the Cas9 protein set forth in SEQ ID NO: 92 (e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the variant Cas9 protein can still bind to target DNA in a site-specific manner (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA. In some cases, the CRISPR-Cas effector protein of a subject CRISPR-Cas fusion protein is a nickase (e.g., cleaves one strand of a double stranded target nucleic acid but not the other strand) (e.g., the Cas9 protein can be a nickase, e.g., can include one or more amino acid mutations that make it a nickase). For example, in some cases, a Cas9 protein of a subject CRISPR-Cas fusion protein has a mutation in a catalytic domain (e.g., a mutation in a RuvC or HNH domain).

[0080] For example, in some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) can cleave the complementary strand of a target nucleic acid but has reduced ability to cleave the non-complementary strand of a target nucleic acid. For example, the Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. Thus, the Cas9 protein can be a nickase that cleaves the complementary strand, but does not cleave the non-complementary strand. As a nonlimiting example, in some cases, a Cas9 protein has a mutation at residue D10 (e.g.,D10A, aspartate to alanine) of SEQ ID NO: 92 (or the corresponding position of any Cas9 protein, e.g., any of the proteins set forth in SEQ ID NOs: 91-126) and can therefore cleave the complementary strand of a double stranded target nucleic acid but has reduced ability to cleave the non-complementary strand of a double stranded target nucleic acid (thus resulting in a single strand break (SSB) instead of a double strand break (DSB) when the variant Cas9 protein cleaves a double stranded target nucleic acid) (see, for example, Jinek et al., Science. 2012 Aug 17;337(6096):816-21). Examples of such amino acid positions in a RuvC domain can include: D10, G12, G17, E762, H982, H983, A984, D986, and / or A987 of the Cas9 protein set forth in SEQ ID NO: 92 (e.g., D10A, G12A, G17A, E762A, H982A, H983A, A984A, and / or D986A).

[0081] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) can cleave the non-complementary strand of a target nucleic acid but has reduced ability to cleave the complementary strand of the target nucleic acid. For example, the Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain. Thus, the Cas9 protein can be a nickase that cleaves the non- complementary strand, but does not cleave the complementary strand (e.g., does not cleave a single stranded target nucleic acid). As a non-limiting example, in some embodiments, the Cas9 protein has a mutation at position H840 (e.g., an H840A mutation, histidine to alanine) of SEQ ID NO: 92 (or the corresponding position of any Cas9 protein, e.g., the Cas9 proteins set forth as SEQ ID NOs: 91-126 and can therefore cleave the non-complementary strand of the target nucleic acid but has reduced ability to cleave (e.g., does not cleave) the complementary strand of the target nucleic acid. Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded target nucleic acid). Examples of such amino acid positions in an HNH domain can include: H840, N854, and / or N863 of the Cas9 protein set forth in SEQ ID NO: 92 (e.g., H840A, N854A, and / or N863A).

[0082] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target nucleic acid. In some cases, the CRIPSR-Cas effector protein is a dead Cas protein (also referred to as catalytically inactive) and thus can be referred to as a dCas (e.g., dCas9, dCas12, and the like). As a non-limiting example, in some cases, a dCas9 protein harbors mutations at residues D10 and H840 (e.g., D10A and H840A) of SEQ ID NO: 92 (or the corresponding residues of another Cas9 protein, e.g., any of the proteins set forth as SEQ ID NOs: 91-126) such that thepolypeptide has a reduced ability to cleave (e.g., does not cleave) both the complementary and the non-complementary strands of a target nucleic acid. Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid. For example, a Cas9 protein of a subject Cas9 fusion polypeptide can have a mutation in one or more of amino acid positions in (i) a RuvC domain: corresponding to D10, G12, G17, E762, H982, H983, A984, D986, and / or A987 of the Cas9 protein set forth in SEQ ID NO: 92 (e.g., D10A, G12A, G17A, E762A, H982A, H983A, A984A, and / or D986A); and one or more of amino acid positions in (ii) an HNH domain: corresponding to H840, N854, and / or N863 of the Cas9 protein set forth in SEQ ID NO: 92 (e.g., H840A, N854A, and / or N863A). In some cases, the CRIPSR- Cas effector protein is a dCas12a protein (see, e.g., Cheng et al., Plant Commun. 2023 Jul 10;4(4): 100601 , which used a fusion protein of dCas12a fused to TadA8e). With respect to Cas12, examples of a single mutation in the nuclease domain, such as D832A or E925A or both (D832A / E925A), can cause it to become catalytically inactive (e.g., dCas12a). Another example is E993A of Cas12a. See, e.g., Kang et al., bioRxiv [Preprint], 2023 May 8:2023.05.08.539911 ; Ciurkot et al., Nucleic Acids Research, Volume 49, Issue 13, 21 July 2021 , Pages 7775-7790; Campa et al., Nat Methods 16, 887-893 (2019).

[0083] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) is a variant. In some cases, such a variant is a high fidelity (HF) protein such as a HF Cas9 protein (also referred to as SpCas9-HF1 or HF1 - and also -HF2, -HF3, -HF4) (e.g., see Kleinstiver et al. (2016) Nature 529:490). For example, amino acids N497, R661 , Q695, and Q926 of the amino acid sequence set forth as (SEQ ID NO: 92) (or the corresponding position of another Cas9 protein, e.g., a protein having the amino acid sequence of any of the sequences set forth as SEQ ID NOs: 91-126) can be substituted, e.g., with alanine. In some cases, a suitable parent Cas9 protein exhibits altered PAM specificity, e.g., in some cases the Cas9 is a VRVRFRR Cas9 (which recognizes an NG PAM instead of NGG; see, e.g., SEQ ID NOs: 26 and 27, where SEQ ID NO: 26 is a subject CRISPR-Cas fusion protein because it is fused to a SpyCatcher protein, see, e.g., FIG. 4) see, e.g., Nishimasu et al., Science. 2018 Sep 21 ;361(6408):1259-1262; and Kleinstiver et al. (2015) Nature 523:481. Additional examples of Cas9 variants that can be used, include, but are not limited to: HiFiCas9 (e.g., R691A), eSpCas9 (e.g., K810A, K1003A, R1060A), eSpCas9 (e.g., D1135E), HypaCas9 (e.g., N692A, M694A, Q695A, H698A), xCas9 (e.g., E108G, S217A,A262T, S409I, E480K, E543D, M694I, E1219V), Sniper-Cas9 (e.g., F539S, M763I, K890N), evoCas9 (e.g., M495V, Y515N, K526E, R661Q), SpartaCas (e.g., D23A, T67L, Y128V, D1251G), LZ3Cas9 (e.g., N690C, T769I, G915M, N980K), miCas9 (e.g., SV40 NLS linker fused with brex27 motif), SuperFi-Cas9 (e.g., Y1010D, Y1013D, Y1016D, V1018D, R1019D, Q1027D, K1031D) (see, e.g., Allemailem et al, Int J Mol Sci. 2023 Apr 11;24(8):7052) [relative to SEQ ID NO: 92],

[0084] In some cases, the CRISPR-Cas effector protein is a Cas9. Examples of Cas9 proteins include, but are not limited to, those of SEQ ID NOs: 25, 27, and 91-126 (note: SEQ ID NOs: 25 and 27 each include a HIS tag, a D10A mutation, and NLSs, see, e.g., FIG. 4, while SEQ ID NOs: 24 and 26, respectively are a repeat of SEQ ID NOs: 25 and 27 but each further includes a SpyCatcher3 sequence). In some cases, the CRISPR-Cas effector protein is a Cas12a. Examples of Cas12a proteins include, but are not limited to, those of SEQ ID NOs: 127-139. In some cases, the CRISPR- Cas effector protein is a Cas13. Examples of Cas13 proteins include, but are not limited to, those of SEQ ID NOs: 140-149.

[0085] In some cases, the Cas9 is an iGeoCas9 (see, e.g., international patent publication WQ2024112479, which is incorporated herein by reference for such disclosure). For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more) identical to iGeoCas9(C) (SEQ ID NO: 124), which is the same as the wild type GeoCas9 of SEQ ID NO: 91, but with the following mutations: E149G, T182I, N206D, P466Q, Q817R, E843K, E884G, and K908R (see, e.g., Chen et al., bioRxiv. Preprint. 2023 Nov 15: doi:10.1101 / 2023.11.15.566339). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more) identical to iGeoCas9(C) (SEQ ID NO: 34).

[0086] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more) identical to iGeoCas9(G)(SEQ ID NO: 125), which is the same as SEQ ID NO:1, but with the following mutations: E149G, T182I, N206D, P466Q, E843K, E884G, K908R, T1015A, and D1017N (see, e.g., Chen et al., bioRxiv. Preprint. 2023 Nov 15: doi:10.1101 / 2023.11.15.566339). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more) identical to iGeoCas9(G)(SEQ ID NO: 125).

[0087] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more) identical to any one of the Cas9 sequences of SEQ ID NOs.: 91-126. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more) identical to any one of the Cas9 sequences of SEQ ID NOs.: 91-126.

[0088] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to the Cas9 sequence of SEQ ID NO: 25 or 27. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to the Cas9 sequence of SEQ ID NO: 25 or 27. In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of SEQ ID NO: 25 or 27.

[0089] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more) identical to any one of the Cas12a sequences of SEQ ID NOs.: 127-139. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more) identical to any one of the Cas12a sequences of SEQ ID NOs.: 127-139.

[0090] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more) identical to any one of the Cas13 sequences of SEQ ID NOs.: 140-149. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more) identical to any one of the Cas13 sequences of SEQ ID NOs.: 140-149.

[0091] For additional information related to programmable gene editing tools (e.g., CRISPR- Cas RNA-guided proteins such as Cas9, CasX, CasY, Cas12a, Cas13, Zinc finger proteins such as Zinc finger nucleases, TALE proteins such as TALENs, CRISPR-Cas guide RNAs, PAMs, and the like) refer to, for example, Dreier, et al., (2001) J Biol Chem 276:29466-78; Dreier, et al., (2000) J Mol Biol 303:489-502; Liu, et al., (2002) J Biol Chem 277:3850-6); Dreier, et al., (2005) J Biol Chem 280:35588-97; Jamieson, etal., (2003) Nature Rev Drug Discov 2:361-8; Durai, et al., (2005) Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus and Carroll, (2005) Nat Biotechnol 23:967-73; Pabo, et al., (2001) Ann Rev Biochem 70:313-40; Wolfe, et al., (2000) Ann Rev Biophys Biomol Struct 29:183-212; Segal and Barbas, (2001) Curr Opin Biotechnol 12:632-7; Segal, et al., (2003) Biochemistry 42:2137-48; Beerli and Barbas, (2002) Nat Biotechnol 20:135-41; Carroll, et al., (2006) Nature Protocols 1 :1329; Ordiz, et al., (2002) Proc Natl Acad Sci USA 99:13290-5; Guan, et al., (2002) Proc Natl Acad Sci USA 99:13296-301 ; Sanjana et al., Nature Protocols, 7:171-192 (2012); Zetsche et al, Cell. 2015 Oct 22;163(3):759-71 ; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385- 97; Jinek et al., Science. 2012 Aug 17;337(6096):816-21 ; Chylinski et al., RNA Biol. 2013 May; 10(5): 726-37; Ma et al., Biomed Res Int. 2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15644-9; Jinek et al., Elife.2013;2:e00471 ; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al, Cell. 2013 Feb 28; 152(5): 1173-83; Wang et al., Cell. 2013 May 9;153(4):910-8; Auer et. al., Genome Res. 2013 Oct 31 ; Chen et. al., Nucleic Acids Res. 2013 Nov 1 ;41 (20):e19; Cheng et. al., Cell Res. 2013 Oct;23(10):1163-71 ; Cho et. al., Genetics. 2013 Nov; 195(3): 1177-80; DiCarlo et al., Nucleic Acids Res. 2013 Apr;41 (7):4336-43; Dickinson et. al., Nat Methods. 2013 Oct; 10(10): 1028-34; Ebina et. al., Sci Rep. 2013;3:2510; Fujii et. al, Nucleic Acids Res. 2013 Nov 1 ;41(20):e187; Hu et. al., Cell Res. 2013 Nov;23(11):1322-5; Jiang et. al., Nucleic Acids Res. 2013 Nov 1 ;41(20):e188; Larson et. al., Nat Protoc. 2013 Nov;8(11):2180-96; Mali et. at., Nat Methods. 2013 Oct;10(10):957-63; Nakayama et. al., Genesis. 2013 Dec;51(12):835- 43; Ran et. al., Nat Protoc. 2013 Nov;8(11):2281-308; Ran et. al., Cell. 2013 Sep 12; 154(6): 1380-9; Upadhyay et. al., G3 (Bethesda). 2013 Dec 9;3(12):2233-8; Walsh et. al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15514-5; Xie et. al., Mol Plant.2013 Oct 9; Yang et. al., Cell. 2013 Sep 12; 154(6): 1370-9; Briner et al., Mol Cell.2014 Oct 23;56(2):333-9; Burstein et al., Nature. 2016 Dec 22 - Epub ahead of print; Gao et al., Nat Biotechnol. 2016 Jul 34(7):768-73; Shmakov et al., Nat Rev Microbiol. 2017 Mar; 15(3): 169-182; Makarova et al., Nat Rev Microbiol. 2020 Feb;18(2):67-83; as well as international patent application publication Nos. W02002099084; WOOO / 42219; WO02 / 42459; W02003062455; W003 / 080809; W005 / 014791; W005 / 084190; W008 / 021207; W009 / 042186; WO09 / 054985; and W010 / 065123; U.S. patent application publication Nos. 20030059767, 20030108880, 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919;20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; 20140377868; 20150166983; and 20160208243; and U.S. Patent Nos. 6,140,466; 6,511 ,808; 6,453,242 8,685,737; 8,906,616; 8,895,308; 8,889,418;8,889,356; 8,871 ,445; 8,865,406; 8,795,965; 8,771 ,945; and 8,697,359; all of which are hereby incorporated by reference in their entirety.

[0092] In some cases, the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas9 polypeptide. In some cases, the Cas9 polypeptide is a Streptococcus pyogenes Cas9 (spyCas9) polypeptide. In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the Cas9 polypeptide is a Geobacillus thermodenitrificans Cas9 (GeoCas9) polypeptide. In some cases, the Cas9 polypeptide is a Neisseria meningitidis Cas9 (NmeCas9) polypeptide.

[0093] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 51 or 52 (e.g., 51). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 51 or 52 (e.g., 51). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to SEQ ID NO: 51 or 52 (e.g., 51). In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of SEQ ID NO: 51 or 52 (e.g., 51).

[0094] In some cases, the CRISPR-Cas fusion protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 24 or 26 (e.g., 24). In some cases, the CRISPR-Cas fusion protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 24 or 26 (e.g., 24). In some cases, theCRISPR-Cas fusion protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to SEQ ID NO: 24 or 26 (e.g., 24). In some cases, the CRISPR-Cas fusion protein comprises the amino acid sequence of SEQ ID NO: 24 or 26 (e.g., 24).

[0095] As illustrative examples, in some cases, the first member of the split-protein binding pair (e.g., catcher protein, partner tag protein, GFP1-10, GFP11) is fused N-terminal to the CRISPR-Cas effector protein (e.g., nCas9). In some cases, the first member of the split-protein binding pair (e.g., catcher protein, partner tag protein, GFP1-10, GFP11) is fused C-terminal to the CRISPR-Cas effector protein (e.g., nCas9). In some cases, a catcher protein (a SpyCatcher protein) is fused N-terminal to the CRISPR-Cas effector protein (e.g., nCas9). In some cases, a catcher protein (a SpyCatcher protein) is fused C-terminal to the CRISPR-Cas effector protein (e.g., nCas9). In some cases, a partner tag protein (e.g., a SpyTag protein) is fused N- terminal to the CRISPR-Cas effector protein (e.g., nCas9). In some cases, a partner tag protein (e.g., a SpyTag protein) is fused C-terminal to the CRISPR-Cas effector protein (e.g., nCas9).PAMs

[0096] A wild type CRISPR-Cas effector protein (e.g., a Cas9 protein, a Cas12 protein) normally has nuclease activity that cleaves a target nucleic acid (e.g., a double stranded DNA (dsDNA)) at a target site defined by the region of complementarity between the guide sequence of the guide RNA and the target nucleic acid. In some cases, site-specific targeting to the target nucleic acid occurs at locations determined by both (i) base-pairing complementarity between the guide nucleic acid and the target nucleic acid; and (ii) a short motif referred to as the “protospacer adjacent motif” (PAM) in the target nucleic acid. For example, when a Cas9 protein binds to (in some cases cleaves) a dsDNA target nucleic acid, the PAM sequence that is recognized (bound) by the Cas9 protein is present on the non-complementary strand (the strand that does not hybridize with the targeting segment of the guide nucleic acid) of the target DNA. For Cas9, the PAM is immediately 3’ of the target sequence (i.e., is positioned downstream of the targeted sequence). In some cases, a PAM sequence has a length in a range of from 1 nt to 15 nt (e.g., 1 nt to 14 nt, 1 nt to 13 nt, 1 nt to 12 nt, 1 nt to 11 nt, 1 nt to 10 nt, 1 nt to 9 nt, 1 nt to 9 nt, 1 nt to 8 nt, 1 nt to 7 nt, 1 nt to 6 nt, 1 nt to 5 nt, 1 nt to 4 nt, 1 nt to 3 nt, 2 nt to 15 nt, 2 nt to 14 nt, 2 nt to 13 nt, 2 nt to12 nt, 2 nt to 11 nt, 2 nt to 10 nt, 2 nt to 9 nt, 2 nt to 8 nt, 2 nt to 7 nt, 2 nt to 6 nt, 2 nt to 5 nt, 2 nt to 4 nt, 2 nt to 3 nt, 2 nt, or 3 nt).

[0097] CRISPR-Cas effector proteins (e.g., Cas9) from different species can have different PAM sequence requirements. For example, in some embodiments (e.g., when the Cas9 protein is derived from S. pyogenes or a closely related Cas9 is used; see for example, Chylinski et al., RNA Biol. 2013 May;10(5):726-37; and Jinek et al., Science. 2012 Aug 17;337(6096):816-21 ; both of which are hereby incorporated by reference in their entirety), the PAM sequence can be NRG because the S. pyogenes Cas9 PAM (PAM sequence) is NAG or NGG (or NRG where “R” is A or G). For example, a Cas9 PAM sequence for S. pyogenes Cas9 can be: NGG, NAG, AGG, CGG, GGG, TGG, AAG, CAG, GAG, and TAG. In some cases, the PAM is NGG.

[0098] In some cases (e.g., when a Cas9 protein is derived from the Cas9 protein of Neisseria meningitidis or a closely related Cas9 is used), the PAM sequence (e.g., of a target nucleic acid) can be 5’-NNNNGANN-3’, 5’-NNNNGTTN-3’, 5’-NNNNGNNT-3’, 5’-NNNNGTNN-3’, 5’-NNNNGNTN-3’, or 5’-NNNNGATT-3’, where N is any nucleotide. In some embodiments (e.g., when a Cas9 protein is derived from Streptococcus thermophilus #1 or a closely related Cas9 is used), the PAM sequence (e.g., of a target nucleic acid) can be 5’-NNAGAA-3’, 5’-NNAGGA-3’, 5’-NNGGAA-3’, 5’-NNANAA-3’, or 5’-NNGGGA-3’ where N is any nucleotide. In some embodiments (e.g., when a Cas9 protein is derived from Treponema denticola (TD) or a closely related Cas9 is used), the PAM sequence (e.g., of a target nucleic acid) can be 5’-NAAAAN-3’, 5’-NAAAAC-3’, 5’-NAAANC-3’, 5’-NANAAC-3’, or 5’-NNAAAC-3’, where N is any nucleotide. As would be known by one of ordinary skill in the art, additional PAM sequences for other Cas9 proteins and other CRISPR-Cas effector proteins are known in the art and / or can readily be determined using bioinformatic analysis (e.g., analysis of genomic sequencing data), and / or routine experimentation. See Esvelt et al., Nat Methods. 2013 Nov;10(11):1116-21 , for additional information. In some cases, the PAM sequence for S. aureus Cas9 is 5’-NNGRR(N)-3’ (in some cases 5’-NNGRRT-3’).

[0099] For Cas12a proteins, the PAM is immediately 5’ of the target sequence (i.e., is positioned upstream of the targeted sequence) (5’ of the non-complementary strand of the target DNA, where the complementary strand hybridizes to the guide sequence of the guide RNA while the non-complementary strand does not directly hybridize with the guide RNA). For example, the wild type protein of SEQ ID NO: 1 (LbCas12a) hasa PAM preference of 5’-TTTV-3’, where V is A, C, or G. As such, LbCas12a can utilize the following PAMs: 5’-TTTA-3’, 5’-TTTC-3’, and 5’-TTTG-3’.

[0100] Example PAMs for wild type Cas12a proteinsPAM: 5 -TTTV-3’ : LbCas12a (statistically calculated from experimental results) PAM: 5’-TTTV-3’ : AsCas12aPAM: 5’-TTN-3’ : FnCas12a, PmCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, TsCas12a, and BsCas12a*where N=(A, C, G, or T); V=(A, C, or G); Y=(C or T); K=(G or T); S=(C or G)Deaminases

[0101] A subject programmable base editor includes a deaminase protein (i.e., a deaminase). The deaminase protein can be any convenient deaminase, and many will be known to one of ordinary skill in the art. For example, the deaminase can be any deaminase used in base editors (where a deaminase is translationally fused to a CRISPR-Cas protein). Examples of base editing enzymes (deaminase proteins (deaminases)) include, but are not necessarily limited to those used in: cytosine base editors (CBEs), adenine base editors (ABEs), cytosine-to-guanine base editors (CGBEs), and simultaneous adenine and cytosine base editors (ACBEs). The deaminase - be it a cytosine deaminase (e.g. APOBEC) or an adenosine deaminase (e.g. engineered TadA) - will dictate whether the base editor is a CBE or an ABE. CBEs contain cytosine deaminases, while ABEs typically contain adenine deaminases (though several groups have recently engineered CBEs derived from the conventional TadA adenosine deaminase domain). Thus, examples of deaminase proteins (deaminases) include, e.g., an adenosine deaminase (e.g., a TadA deaminase such as TadA-8e); a cytidine deaminase (e.g., an activation-induced cytidine deaminase (AID); APOBEC such as APOBEC3G; and the like); a guanine deaminase, a simultaneous adenine and cytosine deaminase; and the like.

[0102] Base editors include cytidine base editors (CBEs) (also referred to as cytosine base editors), which use a cytidine deaminase (e.g., an activation-induced cytidine deaminase / apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID / APOBEC) protein family deaminase) fused to a gene editing protein such as a CRISPR-Cas effector (e.g., nCas9). Base editors also include adenosine base editors (ABEs) (also referred to as adenine base editors), which use an adenosine deaminase (e.g., a tRNA adenosine deaminase (TadA) protein such as TadA-8e) fused to a gene editing protein such as a CRISPR-Cas effector (e.g., nCas9).

[0103] CBEs deaminate cytidine to uridine (subsequently replaced with thymidine by cellular DNA repair), producing C-to-T mutations along the forward strand and complementary G-to-A mutations along the reverse strand. ABEs deaminate adenosine to inosine (subsequently replaced with guanosine), producing A-to-G mutations along the forward strand and T-to-C mutations along the reverse strand.

[0104] In some cases, an adenine deaminase is a TadA deaminase. In some cases, the TadA deaminase is TadA-8e (ABEs that use TadA-8e are sometimes referred to as ABE8e) (see, e.g., Richter at al., Nat Biotechnol. 2020 Jul;38(7):883-891). Variants of ABE8e have also been identified (e.g., NG-ABE9e, see e.g., Tu et al., Mol Ther. 2022 Sep 7;30(9):2933-2941), and such variants can also be used. In some cases, TadA- 8e can be repurposed for cytosine conversion (see, e.g., Chen at al., Nat Biotechnol 41 , 663-672 (2023)). For example, introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C G-to-G C editing. Likewise, mutations at N46 and Y73 in TadA can prevent A«T-to-G«C editing and improve C*G-to-T«A editing with expanded sequence-context compatibility, respectively (see, e.g., Zhang et al., Nat Commun. 2024 Feb 24;15(1):1697). Thus, cytosine base editors (CBEs) can be TadA-derived, and can be referred to as TadCBEs. For additional information related to deaminases, see, e.g., Komor et al, Nature 533.7603 (2016): 420-424; Gaudelli et al., Nature 551 , 464-471 (2017); and US patent application publications US20170121693 and US20220170027, all of which are incorporated by reference herein in their entirety.

[0105] In some embodiments, the subject deaminase protein is an adenine deaminase (e.g., TadA-8e). In some embodiments, the subject deaminase protein is a cytosine deaminase (e.g., an AID / APOBEC family member, a variant of TadA, e.g., a variant of TadA-8e, that acts as a cytosine deaminase, e.g., Td-CGBE, and the like). In some cases in which the deaminase is a cytosine deaminase, the CRISPR-Cas effector protein (e.g., nCas9) is also fused to a uracil glycosylase inhibitor (UGI) domain (in addition to being fused to a member of a split-protein binding pair) (see, e.g., Example 4, FIG. 4, and SEQ ID NO: 73).

[0106] Examples of deaminases include, but are not limited to:>TadA-8e:SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLSDFYRMPRQVFNA QKKAQSSIN (SEQ ID NO: 74)>TadA-8e (plus N-terminal linker fusion):SETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF EPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGIL ADECAALLSDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 32)>TadA-8e-QT:SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRQSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALTSDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 75)>TadA-8e-QT (plus N-terminal linker fusion):SETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF EPCVMCAGAMIHSRIGRVVFGVRQSKRGAAGSLMNVLNYPGMNHRVEITEGIL ADECAALTSDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 34)>TadA-8e_TTISEVEFTHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHD PTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGV RNSKRGATGSLMNVLNYPGMNHRVEITEGILADECAALLSDIYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 35)>TadA-8e-CD (cytoxx deaminase)SEVEFSHEYWMRHALTLAKRARDEGEAPVGAVLVLNNRVIGEGWVRRIGLHD PTAHAEIMALRQGGLVMQNPRLIDATLYVTFEPCVMCAGAMINSRIGRWFGV RNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINS (SEQ ID NO: 33)

[0107] Further examples include those of SEQ ID NOs: 28-31.

[0108] In some cases, the deaminase is an adenosine deaminase, in some cases capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.

[0109] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTG AAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 55).

[0110] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following amino acid sequence: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGW NRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRV VFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQ KKAQSSTD (SEQ ID NO: 56).

[0111] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Staphylococcus aureus adA amino acid sequence: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAE HIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGS LMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN: (SEQ ID NO: 57).

[0112] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Bacillus subtilis TadA amino acid sequence: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVI DEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLM NLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE (SEQ ID NO: 58).

[0113] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Salmonella typhimurium TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGW NRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLI DVLH H PGM N H RVEI I EGVLRDECATLLSDFFRM RRQEI KALK KADRAEGAGPAV (SEQ ID NO: 59).

[0114] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Shewanella putrefaciens TadA amino acid sequence: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILC LRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARWYGARDEKTGAAGTWN LLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 60).

[0115] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Haemophilus influenzae F3031 TadA amino acid sequence: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSD PTAHAEI I ALRNGAKN IQNYRLLNSTLYVTLEPCTMCAGAI LHSRI KRLVFGASDYKTG AIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK (SEQ ID NO: 61).

[0116] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Caulobacter crescentus TadA amino acid sequence: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAH DPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDP KGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 62).

[0117] In some cases, an adenosine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following Geobacter sulfurreducens TadA amino acid sequence: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSND PSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPK GGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFI DERKVPPEP (SEQ ID NO: 63).

[0118] In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation induced deaminase (AID).

[0119] In some cases, a cytidine deaminase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following amino acid sequence: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGC HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTA RLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHE NSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 64).

[0120] In some cases, a cytidine deaminase is an AID and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following amino acid sequence: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGC HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTA RLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKENHERTFKAWEGLHENSVRLSRQLR RILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 65).

[0121] In some cases, a cytidine deaminase is an AID and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to the following amino acid sequence: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGC HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTA RLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHE NSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 66).

[0122] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-35 and 55- 66. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-35 and 55-66. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-35 and 55-66. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 28-35 and 55-66.

[0123] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-35. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-35. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-35. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 28-35.

[0124] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-31. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-31. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 28-31. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 28-31.

[0125] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32-35. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32-35. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32-35. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 32-35.

[0126] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32-35 and 74- 75. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32-35 and 74-75. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32-35 and 74-75. In some cases,the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 32-35 and 74-75.

[0127] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 33, 35, and 74- 75. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 33, 35, and 74-75. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 33, 35, and 74-75. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 33, 35, and 74-75.

[0128] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 35, and 74-75. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 35, and 74-75. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 35, and 74-75. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 35, and 74-75.

[0129] In some cases, the deaminase protein comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32 and 34-35. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32 and 34-35. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 32 and 34-35. In some cases, the deaminase protein comprises the amino acid sequence of any one of SEQ ID NOs: 32 and 34-35.

[0130] In some cases, the deaminase protein (adenosine deaminase) comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO:32. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 32. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to SEQ ID NO: 32. In some cases, the deaminase protein comprises the amino acid sequence of SEQ ID NO: 32.

[0131] In some cases, the deaminase protein (adenosine deaminase) comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 28. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 28. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to SEQ ID NO: 28. In some cases, the deaminase protein comprises the amino acid sequence of SEQ ID NO: 28.

[0132] In some cases, the deaminase protein (cytidine deaminase) comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO:33. In some cases, the deaminase protein comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 33. In some cases, the deaminase protein comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to SEQ ID NO: 33. In some cases, the deaminase protein comprises the amino acid sequence of SEQ ID NO: 33.

[0133] In some embodiments, the deaminase protein is a deaminase that acts on RNA, e.g., is an RNA deaminase, e.g., including A to I and / or C to U editing enzymes. For example, in some cases the deaminase protein is ADAR (adenosine deaminase acting on RNA).

[0134] In some cases, the second member of the split-protein binding pair (e.g., catcher protein, partner tag protein, GFP1-10, GFP11) is fused C-terminal to the deaminase (e.g., adenosine deaminase such as TadA-8e). In some cases, the second member of the split-protein binding pair (e.g., catcher protein, partner tag protein, GFP1-10, GFP11) is fused C-terminal to the deaminase (e.g., adenosine deaminase such as TadA-8e). In some cases, a partner tag protein (a SpyTag) is fused C-terminal to the deaminase (e.g., adenosine deaminase such as TadA-8e). In some cases, a catcherprotein (e.g., a SpyCatcher protein) is fused N-terminal to the deaminase (e.g., adenosine deaminase such as TadA-8e).

[0135] Prior structural studies have shown that TadA deaminase domains, when expressed as monomers, can have a natural tendency to form intermolecular dimers. This dimerization behavior can possibly lead to the formation of multimeric base editor (BE) RNPs, which may in some scenarios hinder genomic search efficiency and / or increase the risk of aggregation. To circumvent these potential liabilities, in some cases, a subject deaminase fusion protein includes two deaminase regions (e.g., in some cases linked via a flexible linker) - in some cases they can be the same, e.g., both TadA-8e, and in some cases they can be different. This double deaminase architecture can result in a pre-formed dimeric deaminase unit, thus promoting / favoring intramolecular dimer formation (e.g., intramolecular TadA dimer formation), over intermolecular dimerization.Nuclear localization signals (NLSs)

[0136] In some embodiments, the deaminase fusion protein (e.g., TadA-8e fusion) and / or the CRISPR-Cas fusion protein (e.g., nCas9 fusion) includes one or more nuclear localization signals (NLSs). Examples of NLSs will be known to one of ordinary skill in the art and any convenient NLS can be used. Examples of NLSs include, but are not limited to those listed in Table 1. Examples of NLSs include, but are not limited to: nucleoplasmin NLS (KRPAATKKAGQAKKKK; SEQ ID NO: 36), SV40 NLS (PKKKRKV; SEQ ID NO: 37), cmyc NLS (PAAKKKKLD; SEQ ID NO: 38), and BP- SV40 NLS (KRTADGSEFEPKKKRKV; SEQ ID NO: 39). In some cases, the deaminase protein includes one or more BP-SV40 NLSs.

[0137] Additional examples include, but are not limited to: RQRRNELKRSP (SEQ ID NO: 40); the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 41); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 42) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 43) and PPKKARED (SEQ ID NO: 44) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 45) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 46) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 47) and PKQKKRK (SEQ ID NO: 48) of the influenza virus NS1 ; the sequence RKLKKKIKKL (SEQ ID NO: 49) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 50) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK(SEQ ID NO: 67) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 68) of the steroid hormone receptors (human) glucocorticoid. For additional NLS sequences see, e.g., Lu et al., Cell Commun Signal. 2021 May 22;19(1):60; and Kosugi et al., J Biol Chem. 2009 Jan 2;284(1):478-485.

[0138] In some cases, the deaminase fusion protein (e.g., TadA-8e fusion) and / or the CRISPR-Cas fusion protein (e.g., nCas9 fusion) includes one or more linker-NLS1- linker-NLS2-linker (hiNLS) modules. In some cases, the first nuclear localization signal (NLS), referred to herein as NLS1 , and the second NLS, referred to herein as NLS2, are the same sequence. In such cases, the hiNLS module can be referred to as linker-NLS-linker-NLS-linker. In some cases, the first and second NLSs (i.e., NLS1 and NLS2) are not the same - they are different from one another, i.e., they differ by amino acid sequence.

[0139] An NLS (NLS1 and / or NLS2) can be any convenient NLS. Any convenient NLS can be used for NLS1, just as any convenient NLS can be used for NLS2. Examples of NLSs include, but are not limited to, those listed in Table 1. In some embodiments, NLS1 and NLS2 are each independently selected from Table 1.

[0140] As a non-limiting illustrative example, in some cases, NLS1 and NLS2 are different, NLS1 is PKKKRKV (SEQ ID NO: 37), and NLS2 is selected from Table 1. Likewise, in some cases, NLS1 and NLS2 are different, NLS2 is PKKKRKV (SEQ ID NO: 37), and NLS1 is selected from Table 1. In some cases, NLS1 and NLS2 are different, NLS1 is PAAKKKKLD (SEQ ID NO: 38), and NLS2 is selected from Table 1. Likewise, in some cases, NLS1 and NLS2 are different, NLS2 is PAAKKKKLD (SEQ ID NO: 38), and NLS1 is selected from Table 1.

[0141] In some embodiments, NLS1 and NLS2 are the same NLS, which is selected from Table 1. For example, in some embodiments, NLS1 and NLS2 are PKKKRKV (SEQ ID NO: 37). In some cases, NLS1 and NLS2 are PAAKKKKLD (SEQ ID NO: 38). In some embodiments, NLS1 and NLS2 are KRPAATKKAGQAKKKK (SEQ ID NO: 36). In some cases, NLS1 and NLS2 are PAAKRVKLD (SEQ ID NO: 69). In some embodiments, NLS1 and NLS2 are KRTADGSEFESPKKKRKVE (SEQ ID NO: 70). In some cases, NLS1 and NLS2 are KRTADGSEFESPKKARKVE (SEQ ID NO: 71). In some embodiments, NLS1 and NLS2 are KRTADGSEFESPKKKAKVE (SEQ ID NO: 72). In some cases, NLS1 and NLS2 are KR(X1)5-I5KK(X2)(X3)KV (SEQ ID NO: xx), where X1 can be any amino acid, the “5-15” denotes a string of from 5-15 X1 residues, X2 is lysine or alanine, and X3 is lysine, arginine, or alanine.

[0142] Table 1. Example NLS Amino acid sequences. Linker sequences are italicized.Notes:• BP-SV40C is a BP-SV40 bipartite NLS consensus sequence. X1 can be any amino acid, and the “5-15” denotes a string of from 5-15 X residues. In some cases, X2 is lysine or alanine. In some cases, X3 is lysine, arginine, or alanine.• Basic charges (R,H,K): SV40 - 5; c-Myc - 4; NP - 8• [ADE] is any amino acid except Asp or Glu

[0143] SEQ ID NOs: 16-23 provide examples of deaminase proteins that include NLSs (SEQ ID NOs: 16, 18, 20, and 22 are deaminase fusion proteins because they are fused to a SpyTag protein; see, e.g., FIG. 4). As such, in some cases, the deaminase protein (or deaminase fusion protein) comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 16-23. In some cases, the deaminase protein (or deaminase fusion protein) comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 16-23. In some cases, the deaminase protein (or deaminase fusion protein) comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one ofSEQ ID NOs: 16-23. In some cases, the deaminase protein (or deaminase fusion protein) comprises the amino acid sequence of any one of SEQ ID NOs: 16-23.

[0144] SEQ ID NOs: 24-27 and 53-54 provide examples of CRISPR-Cas effector proteins (nCas9 proteins with a D10A mutation) that include NLSs (SEQ ID NOs: 24 and 26 are CRISPR-Cas fusion proteins because they are fused to a SpyCatcher protein; see, e.g., FIG. 4). As such, in some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 24-27 and 53-54. In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 24-27 and 53-54. In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to any one of SEQ ID NOs: 24-27 and 53-54. In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises the amino acid sequence of any one of SEQ ID NOs: 24-27 and 53-54.

[0145] In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises an amino acid sequence that is 80% or more (85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 53 or 54 (e.g., 53). In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises an amino acid sequence that is 90% or more (95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to SEQ ID NO: 53 or 54 (e.g., 53). In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises an amino acid sequence that is 95% or more (98% or more, 99% or more, or 100%) identical to SEQ ID NO: 53 or 54 (e.g., 53). In some cases, the CRISPR-Cas effector protein (or CRISPR-Cas fusion protein) comprises the amino acid sequence of SEQ ID NO: 53 or 54 (e.g., 53).Linkers

[0146] Linkers can be used to connect the various components within a given fusion protein (see, e.g., FIG. 4). In some cases in which an hiNLS module is used, each of the three linkers of an hiNLS module (which can be referred to as linkerl , Iinker2, and linkers - for example: Iinker1-NLS1-Iinker2-NLS2-Iinker3) can be the same or different from one another. In some cases, all three of the linkers are the same. In some cases,two of the linkers are the same (where the different linker may be in position linkerl , linker or Iinker3). In some cases, the 3 linkers are different from one another.Flexible amino acid linkers will be known to one of ordinary skill in the art and any convenient linker can be used. Examples of linkers that can be used (e.g., in some cases as part of an hiNLS module) include, but are not necessarily limited to: glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 82), GGSGGSn (SEQ ID NO: 83), and GGGSn (SEQ ID NO: 84), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. Example linkers can comprise amino acid sequences such as: GGSG (SEQ ID NO: 77), GGSGG (SEQ ID NO: 78), GSGSG (SEQ ID NO: 76), GSGGG (SEQ ID NO: 79), GGGSG (SEQ ID NO: 80), GSSSG (SEQ ID NO: 81), and the like. As such, in some cases, all three of the linkers are independently selected from GGSG (SEQ ID NO: 77), GGSGG (SEQ ID NO: 78), GSGSG (SEQ ID NO: 76), GSGGG (SEQ ID NO: 79), GGGSG (SEQ ID NO: 80), and GSSSG (SEQ ID NO: 81).

[0147] In some cases, at least one of the linkers of a given hiNLS module is GSGSG (SEQ ID NO: 76). In some cases, at least two of the linkers of a given hiNLS module are GSGSG (SEQ ID NO: 76). In some cases, all 3 of the linkers of a given hiNLS module are GSGSG (SEQ ID NO: 76). See, e.g., the example hiNLS module sequences of Table 1.

[0148] In some cases, at least one of the linkers of a given hiNLS module is GGSG (SEQ ID NO: 77). In some cases, at least two of the linkers of a given hiNLS module are GGSG (SEQ ID NO: 77). In some cases, all 3 of the linkers of a given hiNLS module are GGSG (SEQ ID NO: 77).

[0149] In some cases, at least one of the linkers of a given hiNLS module is GGSGG (SEQ ID NO: 78). In some cases, at least two of the linkers of a given hiNLS module are GGSGG (SEQ ID NO: 78). In some cases, all 3 of the linkers of a given hiNLS module are GGSGG (SEQ ID NO: 78).

[0150] In some cases, at least one of the linkers of a given hiNLS module is GSGGG (SEQ ID NO: 79). In some cases, at least two of the linkers of a given hiNLS module are GSGGG (SEQ ID NO: 79). In some cases, all 3 of the linkers of a given hiNLS module are GSGGG (SEQ ID NO: 79).

[0151] In some cases, at least one of the linkers of a given hiNLS module is GGGSG (SEQ ID NO: 80). In some cases, at least two of the linkers of a given hiNLS module are GGGSG (SEQ ID NO: 80). In some cases, all 3 of the linkers of a given hiNLS module are GGGSG (SEQ ID NO: 80).

[0152] In some cases, at least one of the linkers of a given hiNLS module is GSSSG (SEQ ID NO: 81). In some cases, at least two of the linkers of a given hiNLS module are GSSSG (SEQ ID NO: 81). In some cases, all 3 of the linkers of a given hiNLS module are GSSSG (SEQ ID NO: 81).Nucleic Acids

[0153] Provided are nucleic acid systems, which, e.g., include one or more nucleic acids. In some cases, a subject nucleic acid system includes one nucleic acid (e.g., an expression construct such as a plasmid for viral vector). In some cases, a subject nucleic acid system includes a nucleic acid that encodes (i.e. , includes a nucleotide sequence that encodes) a subject CRISPR-Cas fusion protein. In some cases, a subject nucleic acid system includes a nucleic acid that encodes (i.e., includes a nucleotide sequence that encodes) a subject deaminase fusion protein. In some cases, a subject nucleic acid system includes a nucleic acid that encodes both a subject CRISPR-Cas fusion protein and a subject deaminase fusion protein (e.g., in some cases where each fusion protein is under the control of a different promoter, such as two different inducible promoters so that expression of each fusion protein can be accomplished while the other fusion protein is not being expressed or is being expressed at very low levels). In some cases, a subject nucleic acid system includes more than one nucleic acid (e.g., includes two nucleic acids), where one encodes a subject CRISPR-Cas fusion protein, and the other encodes (i.e., includes a nucleotide sequence that encodes) a subject deaminase fusion protein.

[0154] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a protein of the present disclosure (e.g., a CRISPR-Cas fusion protein, a deaminase fusion protein). The present disclosure provides a nucleic acid / protein complex comprising: a) a subject programmable base editor; and b) a guide RNA.

[0155] The present disclosure provides one or more nucleic acids (e.g., RNA and / or DNA) comprising one or more of: a nucleotide sequence encoding a CRISPR-Cas fusion protein, a nucleotide sequence encoding a deaminase fusion protein, a guide RNA (which can include two separate nucleotide sequences in the case of dual guide RNA format or which can include a single nucleotide sequence in the case of single guide RNA format), and a nucleotide sequence encoding a guide RNA.

[0156] The present disclosure provides a nucleic acid (e.g., an mRNA, a plasmid, a viral vector, a minicircle DNA) comprising a nucleotide sequence encoding a CRISPR-Cas fusion protein. The present disclosure provides a nucleic acid (e.g., an mRNA, aplasmid, a viral vector, a minicircle DNA) comprising a nucleotide sequence encoding a deaminase fusion protein. The present disclosure provides a recombinant expression vector (e.g., viral DNA, plasmid DNA, minicircle DNA) that comprises a nucleotide sequence encoding a subject CRISPR-Cas fusion protein. The present disclosure provides a recombinant expression vector (e.g., viral DNA, plasmid DNA, minicircle DNA) that comprises a nucleotide sequence encoding a subject deaminase fusion protein. In some cases, the nucleotide sequence encoding the protein of the present disclosure (e.g., CRISPR-Cas fusion protein and / or deaminase fusion protein) and / or the nucleotide sequence encoding the Cas9 guide RNA is operably linked to a promoter that is operable in a cell type of choice (e.g., a prokaryotic cell such as an E. coli cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell, etc.).

[0157] In some embodiments, a nucleotide sequence encoding a protein of the present disclosure (e.g., a deaminase fusion protein and / or a CRISPR-Cas effector protein) is codon optimized. This type of optimization can entail a mutation of a protein-coding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein (e.g., using commercially or freely available software). Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized protein-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon-optimized protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were an insect cell, then an insect codon-optimized protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a prokaryotic cell such as a bacterial cell (e.g., E. coli), then a bacterial (e.g., E. coli) codon-optimized proteinencoding nucleotide sequence could be generated. In some cases, the nucleotide sequence encoding the CRISPR-Cas fusion protein, and / or the nucleotide sequence encoding the deaminase fusion protein is codon optimized for expression in a prokaryotic cell. In some cases, the nucleotide sequence encoding the CRISPR-Cas fusion protein, and / or the nucleotide sequence encoding the deaminase fusion protein is codon optimized for expression in an E. coli cell. In some cases, the nucleotide sequence encoding the CRISPR-Cas fusion protein, and / or the nucleotide sequenceencoding the deaminase fusion protein is codon optimized for expression in a eukaryotic cell. In some cases, the nucleotide sequence encoding the CRISPR-Cas fusion protein, and / or the nucleotide sequence encoding the deaminase fusion protein is codon optimized for expression in a mammalian cell.

[0158] Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191 ; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0159] Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[0160] In some embodiments, a nucleotide sequence encoding a guide RNA is operably linked to a control element, e.g., a transcription control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a deaminase fusion protein and / or CRISPR-Cas fusion protein is operably linked to a control element, e.g., a transcription control element, such as a promoter.

[0161] The transcription control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcription control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcription control element (e.g., a promoter) can be functional in prokaryotic cells (e.g., bacteria such as E. coli). As another example, in some cases, the transcription control element (e.g., a promoter) can be functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc.).

[0162] Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EF1a, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, beta actin, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to a subject protein, thus resulting in a chimeric polypeptide.

[0163] In some embodiments, a nucleotide sequence encoding a deaminase fusion protein and / or CRISPR-Cas fusion protein polypeptide is operably linked to an inducible promoter. In some embodiments, a nucleotide sequence encoding a deaminase fusion protein and / or CRISPR-Cas fusion protein fusion polypeptide is operably linked to a constitutive promoter.

[0164] A promoter can be a constitutively active promoter (i.e. , a promoter that is constitutively in an active / ”ON” state), it may be an inducible promoter (i.e., a promoter whose state, active / ”ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcription control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state duringspecific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[0165] Promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), beta actin, EF1-alpha, a rous sarcoma virus (RSV) promoter, and the like. Example Pol III promoters (e.g., for expressing a guide RNA) include, but are not necessarily limited to: a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1 ;31 (17)), a human H1 promoter (H1), and the like.

[0166] In some cases, a nucleotide sequence encoding a guide RNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a Pol III promoter such as a U6 promoter, an enhanced U6 promoter, an H1 promoter, and the like). In some cases, a nucleotide sequence encoding a subject deaminase fusion protein and / or CRISPR-Cas fusion protein is operably linked to a promoter operable in a prokaryotic cell such as a bacterial cell, e.g., E. coli. In some cases, a nucleotide sequence encoding a subject deaminase fusion protein and / or CRISPR-Cas fusion protein is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, a beta-actin promoter, an EF1a promoter, an estrogen receptor-regulated promoter, and the like).

[0167] Examples of inducible promoters include, but are not limited toT7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; estrogen and / or an estrogen analog; IPTG; etc.

[0168] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulatedpromoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (ATC)-responsive promoters and other tetracyclineresponsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat- inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0169] In some cases, the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcription control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell; prokaryotic cell).

[0170] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters,benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0171] In some embodiments, a first nucleotide sequence encoding a subject CRISPR-Cas fusion protein, and a second nucleotide sequence encoding a subject deaminase fusion protein are both present on the same nucleic acid molecule (e.g., a plasmid, a viral vector, and the like). Therefore, in some cases, the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by and expressed from the same nucleic acid molecule (e.g., a plasmid, a viral vector, and the like). In some such cases, the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters. In some cases, the first nucleotide sequence and the second nucleotide sequence are operably linked to different copies of the same promoter (in which case, the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by two different transcribed mRNAs).

[0172] In some cases, the first nucleotide sequence and the second nucleotide sequence are in tandem and are expressed from the same promoter (e.g., an inducible promoter such as a T7 promoter) (such that the transcriptional stop sequence is 3’ of both the first and second nucleotide sequences), each having a translation initiation sequence (e.g., Shine-Dalgarno sequence), such that the RNA transcript is bicistronic (i.e. , both proteins are encoded and translated from the same transcript). In some cases, the sequence encoding the CRISPR-Cas fusion protein is 5’ of the sequence encoding the deaminase fusion protein. In some cases, the sequence encoding the CRISPR- Cas fusion protein is 3’ of the sequence encoding the deaminase fusion protein.

[0173] In some such cases (i.e., where the first nucleotide sequence and the second nucleotide sequence are in tandem and are expressed from the same promoter, where the transcriptional stop sequence is 3’ of both the first and second nucleotide sequences, and where each of the two sequences have a translation initiation sequence such that the RNA transcript is bicistronic), a second promoter can be present between the first nucleotide sequence and the second nucleotide sequence such that both sequences are operably linked to the first (5’ most) promoter, while only the 3’ most sequence is operably linked to the second (3’ most) promoter. As such, expression from the first promoter (the 5’ most promoter) would lead to transcription of a bicistronic RNA (encoding both the CRISPR-Cas fusion protein and the deaminase fusion protein); and expression from the second promoter (thedownstream / 3’ promoter) would lead to transcription only of the 3’ most sequence. See, e.g., Figure 11 as an illustrative example of one such embodiment.

[0174] For example, if the sequence encoding the CRISPR-Cas fusion protein is 5’ of the sequence encoding the deaminase fusion protein, expression from the first promoter would lead to a bicistronic mRNA encoding both proteins, and expression from the second promoter would lead to an mRNA encoding the deaminase fusion protein. In such a case, more of the deaminase fusion protein would ultimately be produced because it would be encoded by and translated from both of the generated mRNAs. Likewise, if the sequence encoding the CRISPR-Cas fusion protein is 3’ of the sequence encoding the deaminase fusion protein, expression from the first promoter would lead to a bicistronic mRNA encoding both proteins, and expression from the second promoter would lead to an mRNA encoding the CRISPR-Cas fusion protein. In such a case, more of the CRISPR-Cas fusion protein would ultimately be produced because it would be encoded by and translated from both of the generated mRNAs. Thus, in such a system, one can increase the ratio of one protein relative to the other by positioning the sequence encoding the protein with a higher desired relative level 3' of the sequence encoding the protein with the lower desired relative level.

[0175] In some cases, the second promoter is a copy of the first promoter (i.e. , it is the same promoter, but used twice). In some such cases, the promoter is an inducible promoter (e.g., T7 promoter). In some cases, the first and second promoters are different promoters (both can be inducible, both can be constitutive, or one can be constitutive while the other is inducible).

[0176] Methods of introducing a nucleic acid into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, nucleofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, lipid nanoparticle mediated delivery, and the like.

[0177] Introducing the recombinant expression vector into cells can occur in vivo or can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant expression vector into a target cell can be carried out in vivo or ex vivo or in vitro. Introducing the recombinant expression vector into a target cell can be carried out in vitro.

[0178] In some embodiments, a subject protein (e g., deaminase fusion protein and / or CRISPR-Cas fusion protein) is provided to a cell as RNA (e.g., as mRNA that is translated into protein within a cell). In some embodiments, a guide RNA is provided as RNA. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA. Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, lipid nanoparticle delivery, etc.).

[0179] Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5(7): e11756, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, TranslT®-mRNA Transfection Kit from Mirus Bio LLC, nucleofection, and the like. See also Beumer et al. (2008) PNAS 105(50): 19821- 19826.

[0180] Vectors may be provided directly to a target host cell. In other words, the cells can be contacted with vectors comprising the subject nucleic acids such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors.

[0181] Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).

[0182] Vectors used for providing the nucleic acids encoding guide RNA and / or a protein of the present disclosure (e.g., deaminase fusion protein and / or CRISPR-Cas fusion protein) to a target host cell can include suitable promoters for driving the expression, that is, transcription activation, of the nucleic acid of interest. In other words, in some cases, the nucleic acid of interest will be operably linked to a promoter. This may include ubiquitously acting promoters, for example, the CMV- -actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline. By transcription activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, more usually by 1000 fold. In addition, vectors used for providing a nucleic acid encoding a guide RNA and / or a protein of the present disclosure (e.g., deaminase fusion protein and / or CRISPR-Cas fusion protein) to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide RNA and / or subject protein.

[0183] A nucleic acid comprising a nucleotide sequence encoding a protein of the present disclosure (e.g., deaminase fusion protein and / or CRISPR-Cas fusion protein), is in some cases a DNA. A nucleic acid comprising a nucleotide sequence encoding a protein of the present disclosure (e.g., deaminase fusion protein and / or CRISPR-Cas fusion protein), is in some cases an RNA. Thus, a protein of the present disclosure (e.g., deaminase fusion protein and / or CRISPR-Cas fusion protein) can be introduced into cells as RNA. Methods of introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method used for the introduction of DNA. A protein of the present disclosure (e.g., deaminase fusion protein and / or CRISPR-Cas fusion protein) may instead be provided to cells as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like. The polypeptide may beformulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.

[0184] Additionally or alternatively, a protein of the present disclosure (e.g., deaminase fusion protein, CRISPR-Cas fusion protein, and / or a programmable base editor) can be introduced as protein into a cell (e.g., in some cases Fused to a polypeptide permeant domain to promote uptake by the cell). In some cases, a subject a programmable base editor is introduced to a cell as an RNP (i.e., pre-complexed with a guide RNA). In some cases, a RNP is delivered using peptide-enabled RNP delivery for CRISPR engineering (PERC) (see, e.g., Foss et al., Nature Biomedical Engineering, 2023: p. 1-14). In some cases, a RNP is delivered using a nanoparticle formulation. In some cases, a RNP is delivered using a lipid nanoparticle formulation.

[0185] A number of permeant domains are known in the art and may be used in the nonintegrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 170). As another example, the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, for example, Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr; 4(2): 87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000 Nov. 21 ; 97(24): 13003-8; published U.S. Patent applications 20030220334; 20030083256; 20030032593; and 20030022831 , herein specifically incorporated by reference for the teachings of translocation peptides and peptoids). The nona-arginine (R9) sequence is one of the more efficient PTDs that have been characterized (Wender et al. 2000; Uemura et al. 2002). The site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site will be determined by routine experimentation.

[0186] A protein of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells, and it may be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using methods known in the art.

[0187] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[0188] Also suitable for inclusion in embodiments of the present disclosure are nucleic acids and proteins of the present disclosure that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0189] A protein of the present disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[0190] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[0191] A protein of the present disclosure may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise 20% or more by weight of the desired product, more usually 75% or more byweight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Thus, in some cases, a protein of the present disclosure (e.g., subject Cas9 fusion polypeptide) is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-desired proteins or other macromolecules, etc.).

[0192] In cases in which two or more different targeting complexes are provided to a target cell (e.g., two different guide RNAs that are complementary to different sequences within the same or different target nucleic acid), the complexes may be provided simultaneously (e.g. as two polypeptides and / or nucleic acids), or delivered simultaneously. Alternatively, they may be provided consecutively, e.g. the targeting complex being provided first, followed by the second targeting complex, etc. or vice versa.Methods of producing a programmable base editor

[0193] The present disclosure provides methods of producing a programmable base editor (any of the subject programmable base editors described herein). Such methods include contacting a subject CRISPR-Cas fusion protein with a subject deaminase fusion protein, thus producing the programmable base editor. Notably, the methods disclosed herein include use of two separate proteins (a CRISPR-Cas fusion protein and a deaminase fusion protein) to produce a base editor as opposed to making a base editor as one single protein (e.g., a single translation product from a single nucleotide sequence). The methods of producing described herein (using two proteins as opposed to one) can facilitate high yield production of a programmable base editor. In some cases, the yield of the programmable base editor is 2X or more (e.g., 3X, 5X, 10X, or more) relative to the yield of producing a base editor from a single translation product (e.g., expressed from single plasmid). In some cases, the yield of the programmable base editor is 5X or more (e.g., 10X, or more) relative to the yield of producing a base editor from a single translation product (e.g., expressed from single plasmid).

[0194] In many embodiments, the contacting of the CRISPR-Cas fusion protein with the deaminase fusion protein will take place in vitro not inside of a cell (also referred to as ‘outside of a cell’). In some cases, the contacting can take place inside of a cell (e.g., a prokaryotic cell such as an E. coli cell or a eukaryotic cell such as a mammalian orinsect cell). In some such cases, the CRISPR-Cas fusion protein and the deaminase fusion protein can be induced separately, e.g., one protein can be expressed first, followed by expression of the other protein. As an example, in some cases, the CRISPR-Cas fusion protein will be expressed inside of a cell (e.g., an E. coli cell) (e.g., under the control of an inducible promoter) (e.g., under conditions tailored to production of the CRISPR-Cas fusion protein), and then the deaminase fusion protein will be expressed inside of the same cell (e.g., under the control of a different inducible promoter) (e.g., under conditions tailored to production of the deaminase fusion protein): both proteins would then contact one another inside of the cell and assemble into a programmable base editor. The programmable base editor could then be extracted (and optionally purified / enriched) from the cell. Use of orthogonal tags (e.g., affinity tags) could prevent contamination of the extract with unassembled components.

[0195] As such, and as noted above in more detail, in some embodiments, a first nucleotide sequence encoding a subject CRISPR-Cas fusion protein, and a second nucleotide sequence encoding a subject deaminase fusion protein are both present on the same nucleic acid molecule (e.g., a plasmid, a viral vector, and the like). In some such cases, the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters. Therefore, in some cases, the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by and expressed from the same nucleic acid molecule (e.g., a plasmid, a viral vector, and the like).

[0196] The contacting will take place under conditions sufficient for binding / assembly of the split-protein binding pair (i.e., binding of the first member of the pair with the second member of the pair, thus resulting in formation of a subject programmable base editor). A range of such conditions (for assembly of first and second members of a split-protein binding pair) will be known to one of ordinary skill in the art, and any convenient conditions can be used. In some cases, the conditions will be sufficient for isopeptide bond formation between a catcher protein and a partner tag protein (i.e., contacting results in covalent linkage of the CRISPR-Cas fusion protein with the deaminase fusion protein via post-translational isopeptide bond formation between the catcher protein and the partner tag protein). In some cases, the conditions will be sufficient for binding / assembly of GFP1-10 with GFP11 .

[0197] In some cases, the contacting takes place at a pH (e.g., buffered at a pH) in a range of from 7-8 (e.g., 7.1-7.9, 7.1-7.7, 7.1-7.6, 7.3-8, 7.3-7.9, 7.3-7.7, 7.3-7.6, 7.4-8, 7.4- 7.9, 7.4-7.7, 7.4-7.6, or about 7.5). For example, in some cases, the contacting takesplace at a pH of about 7.5. In some cases, the contacting takes place at a salt concentration in a range of from 200-400 mM NaCI (e.g., 200-350, 200-325, 200-325, 250-400, 250-350, 250-325, 250-315, 275-400, 275-350, 275-325, 275-315, 285-400, 285-350, 285-325, 285-315, or about 300nM) (e.g., a salt concentration that is too low can in some cases result in precipitation). For example, in some cases, the contacting takes place at a salt concentration of about 300mM NaCI. In some cases, the contacting takes place at a pH in a range of from 7-8 (e.g., 7.1-7.9, 7.1-7.7, 7.1-7.6, 7.3-8, 7.3-7.9, 7.3-7.7, 7.3-7.6, 7.4-8, 7.4-7.0, 7.4-7.7, 7.4-7.6, or about 7.5); and at a salt concentration in a range of from 200-400 mM NaCI (e.g., 200-350, 200-325, 200- 325, 250-400, 250-350, 250-325, 250-315, 275-400, 275-350, 275-325, 275-315, 285- 400, 285-350, 285-325, 285-315, or about 300nM). In some cases, the contacting will take place at a pH of about 7.5 and at a salt concentration of about 300mM NaCI.

[0198] In some embodiments, after the fusion proteins are contacted to form a programmable base editor, the programmable base editor is purified / enriched. Thus, in some cases, a subject method includes (after contacting the CRISPR-Cas fusion protein with the deaminase fusion protein) performing one or more purification steps to enrich for the programmable base editor. In some cases, such purification includes gel filtration (i.e., size exclusion chromatography) (e.g., using a Superdex resin such as a Superdex 200 resin). As an illustrative, non-limiting example, a HiLoad 16 / 60 S200 Superdex column can be used (e.g., using an AKTA system) (see, e.g., the working examples below).Production

[0199] In some embodiments, a subject method includes producing the CRISPR-Cas fusion protein and / or the deaminase fusion protein prior to their contact. In other words, in some cases, a subject method includes contacting the CRISPR-Cas fusion protein with the deaminase fusion protein to produce a subject programmable base editor, while in some cases, a subject method includes producing the CRISPR-Cas fusion protein and / or producing the deaminase fusion protein (e.g., producing both the CRISPR-Cas fusion protein and the deaminase fusion protein), and then placing them in contact to produce the programmable base editor. Thus, in some cases, both fusion proteins are produced as part of the method. Usually, the producing will include expression of the CRISPR-Cas fusion protein and / or the deaminase fusion protein in cells (if both are produced, they will be produced separately in different cells, i.e., they will be produced separately in cells).

[0200] The cells used for production can be any cell suitable for producing a heterologous protein, e.g., for extraction and purification. Both prokaryotic and eukaryotic cells for such purposes (production of a heterologous protein for extraction / purification) will be known to one of ordinary skill in the art, and any convenient cell can be used. For example, a cell used for production can be any of the cells listed elsewhere herein as target cells. In some cases, the cells used for production with be eukaryotic. In some cases, the cells used for production with be prokaryotic. In some cases, the cells used for production will be E. coli.

[0201] After the fusion protein(s) are produced, they generally are extracted from the cells by purification / enrichment methods. General methods for purifying proteins from prokaryotic and eukaryotic cells are known in the art, and any convenient extraction / purification methods can be used. In some cases, the CRISPR-Cas fusion protein is purified / enriched using affinity chromatography (e.g., the fusion protein can include a His-tag and a nickel column can be used). As an illustrative, non-limiting example, a HISTRAP™ FF column prepacked with precharged nickel Sepharose can be used to capture the His-tagged fusion protein. In some cases, the CRISPR-Cas fusion protein is purified / enriched using gel filtration (e.g., using a heparin column). As an illustrative non-limiting example, a HiTrap Heparin HP column can be used (e.g., equilibrated in 20 mM HEPES (pH 7.5), 300 mM NaCI, and 10% (v / v) glycerol, see the working examples below). In some cases, the CRISPR-Cas fusion protein is purified / enriched using both affinity chromatography and gel filtration (e.g., in some cases affinity chromatography followed by gel filtration).

[0202] In some cases, the deaminase fusion protein is purified / enriched using affinity chromatography (e.g., the fusion protein can include a His-tag and a nickel column can be used). As an illustrative non-limiting example, a HISTRAP™ FF column prepacked with precharged nickel Sepharose can be used to capture the His-tagged fusion protein. In some cases, high NaCI (e.g., a range of from 800 mM to 1.2 M) is used in the lysis buffer to, e.g., wash away contaminating nucleic acids that can tend to adhere to the deaminase (e.g., a TadA protein such as TadA-8e).

[0203] In some cases, the purification / enrichment (of the CRISPR-Cas fusion protein and / or the deaminase fusion protein) takes place at a pH (e.g., buffered at a pH) in a range of from 7-8 (e.g., 7.1-7.9, 7.1-7.7, 7.1-7.6, 7.3-8, 7.3-7.9, 7.3-77, 7.3-7.6, 7.4-8, 7.4- 7.9, 7.4-7.7, 7.4-7.6, or about 7.5). For example, in some cases, the purification / enrichment takes place at a pH of about 7.5.Methods of modifying a target nucleic acid

[0204] The present disclosure provides methods of modifying a target nucleic acid (e.g., target DNA) using a subject programmable base editor (any of the subject programmable base editors described herein). Such methods include contacting a target nucleic acid (e.g., a target DNA such as a genomic DNA inside of a cell) with a subject programmable base editor.Target nucleic acids and target cells

[0205] A protein of the present disclosure (e.g., a subject programmable base editor), when bound to a guide RNA, can bind to and modify a target nucleic acid. A target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome, derived from a chromosome, chromosomal, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the guide RNA can hybridize to a target sequence in a target nucleic acid, that target nucleic acid can be targeted - taking the PAM into account for double stranded target nucleic acids, as would be understood by one of ordinary skill in the art and as described elsewhere herein).

[0206] A target nucleic acid can be DNA or RNA. A target nucleic acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). In some cases, a target nucleic acid is a double stranded DNA (dsDNA). In some cases, a target nucleic acid is chromosomal DNA.

[0207] In some cases, the target nucleic acid is a DNA (e.g., in some cases the CRISPR-Cas effector protein is a Cas9 or a Cas12). For example, in some cases the target DNA is genomic DNA, e.g., genomic DNA inside of a cell. In some cases, the target nucleic acid is a RNA, e.g., an mRNA, a miRNA, a tRNA, an rRNA, and the like (e.g., in some cases the CRISPR-Cas effector protein is a Cas13). In some cases, the target sequence of the target nucleic acid (DNA or RNA) is a protein-coding sequence. In some cases, the target sequence of the target nucleic acid (DNA or RNA) is a noncoding sequence (i.e. , does not encode a protein).

[0208] A target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro (e.g., in a cell in culture), inside of a cell in vivo, inside of a cell ex vivo (e.g., a cell recently isolated from an individual); or inside of an organelle (e.g., mitochondrion; nucleus; etc.) within a cell that is in vitro, in vivo, or ex vivo.

[0209] Any cell can be targeted using the methods disclosed herein (e.g., methods of modifying a target nucleic acid). Because the guide RNA provides specificity by hybridizing to target nucleic acid, a cell of interest in the disclosed methods may include a cell from any organism. Examples of target cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; an invertebrate animal cell (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); an insect cell (e.g., a fruit fly, a mosquito; a bee; an agricultural pest; etc.); an arachnid cell (e.g., a spider; a tick; etc.); a vertebrate animal cell (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a mammal cell (e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a lagomorph cell (e.g., a rabbit); an ungulate cell (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a marine mammal cell (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.), and the like.

[0210] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual - in some cases a primary cell, e.g., a primary human cell). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells (a population of cells). A cell can be an animal cell or derived from an animal cell.

[0211] Plant cells include cells of a monocotyledon, and cells of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.

[0212] Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoacell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0213] Target cells can be any convenient cell. In some cases the target cell is a liver, lung, spleen, kidney, or heart cell. In some cases the target cell is a liver or lung cell. In some cases the target cell is a liver cell. In some cases the target cell is a lung cell. Examples of possible target cells include, but are not limited to: a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, an astrocyte, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a liver cell, a lung cell (e.g., a lung epithelial cell), etc. Target cells include stem cells, cancer cells, human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes (e.g., white adipocytes), totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, immune cells (e.g., T cell such as cytotoxic T cell, helper T cell, or regulatory T cell (Treg), B cell, monocyte, natural killer (NK) cell, dendritic cell, macrophage, and the like), mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal muscle cells, islet cells (e.g., beta cells), fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells. Targetcells include, e.g., lung cells, neurons, astrocytes, islet cells, kidney cells, adipocytes, hepatocytes, endothelial cells, muscle cells, cardiomyocytes, retinal cells, and tissueresident stem cells. Target organs and tissues include, e.g., kidney, liver, bone, pancreas, brain, lung, heart, fat, and the like.

[0214] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc ); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.

[0215] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, neurons, astrocytes, islet cells, alpha cells, beta cells delta cells, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells. In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0216] Adult stem cells are resident in differentiated tissue, but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0217] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0218] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer (NK) cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).Delivery

[0219] A protein of the present disclosure (e.g., a subject programmable base editor) can be introduced into (delivered to) a host cell by any of a variety of well-known methods. In some cases, a programmable base editor is delivered with a guide RNA (e.g., in some cases complexed with the guide RNA as an RNP). In some cases, a guide RNA and a programmable base editor are delivered as part of the same composition (e.g., as an RNP), and in some cases they can be delivered separately, as part of separate compositions. A guide RNA can be provided directly (e.g., as one or more RNA molecules), or can be provided as a DNA encoding the guide RNA.

[0220] Methods of introducing a nucleic acid (e.g., guide RNA, mRNA, DNA) into a cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, vertebrate cell, mammalian cell, primate cell, non-human primate cell, human cell, and the like) are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a target cell. Suitable methods include, e.g., viral infection, transfection, nucleofection, conjugation, protoplast fusion, lipofection, nucleofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, agrobacterium-mediated transformation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169- 409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023 ), and the like.

[0221] In some cases, a protein of the present disclosure (e.g., a subject CRISPR-Cas fusion protein and / or a subject deaminase fusion protein) is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes protein. For example, such can be the case when providing such a nucleic acid to a producer cell (i.e., providing to a cell for the purpose of producing the fusion protein(s) and / or producing a subject programmable base editor). In some cases, a protein of the present disclosure (e.g., a subject programmable base editor) is provided to a cell as a protein.

[0222] A protein of the present disclosure (e.g., a subject programmable base editor) can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, a protein of the present disclosure (e.g., a subject programmable base editor) can be injected directly into a cell (e.g., with or without a guide RNA, with or without a nucleic acid encoding a guide RNA). As another example, a preformed complex of a protein of the present disclosure (e.g., a subject programmable base editor) plus a guide RNA is referred to as a ribonucleoprotein (RNP) complex, can be introduced into a cell (e.g., via nucleofection; via injection; via a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to the protein, conjugated to a guide RNA, and the like). As noted above, a guide RNA can be introduced into a cell as RNA or as DNA encoding the RNA (e.g., an expression vector encoding the Cas9 guide RNA).

[0223] In some embodiments, a subject programmable base editor (and in some cases the base editor plus one more guide RNAs) are introduced into a cell using a nanoparticle, and in some cases the nanoparticle is a lipid nanoparticle.

[0224] In some cases, a nanoparticle is used. As used herein, a “nanoparticle” is a particle having at least one dimension in the range of from 1 nm to 1000 nm, from 20 nm to 750 nm, from 50 nm to 500 nm, including 100 nm to 300 nm, e.g., 120-200 nm. The nanoparticle may have any suitable shape, including but not limited to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardropshaped, tetrapod-shaped, prism-shaped, or any other suitable geometric or nongeometric shape. In certain cases, a nanoparticle includes on its surface one or more targeting moieties, e.g., antibodies, ligands, aptamers, small molecules, etc. Nanoparticles include those described in Wang et al. (2010) Pharmacol. Res. 62(2):90-99; Rao et al. (2015) ACS Nano 9(6): 5725-5740; and Byrne et al. (2008) Adv. Drug Deliv. Rev. 60(15):1615-1626.

[0225] In some cases, a lipid nanoparticle is used. As used herein, the term “lipid nanoparticle” (LNP) refers to a transfer vehicle (e.g., for delivering a molecular payload such as nucleic acid and / or protein to a cell) comprising one or more lipids (e.g., ionizable lipids, cationic lipids, non-cationic lipids, neutral lipids, neutral phospholipids, polymerizable lipids, PEG-modified lipids, cholesterol, and the like). The lipid(s) of an LNP can include any convenient lipid. Examples include, but are notlimited to DLin-DMA, DLin-K-DMA, 98NI2-5, CI2-200, DLin-MC3-DMA, DLin-KC2- DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.

[0226] In some cases, an LNP includes a cationic lipid. Cationic lipids typically have a positively charged head group followed by a hydrophobic tail of varying composition. In an aqueous environment, these cationic lipids form micelles with positively charged surfaces that complex with DNA. Examples of cationic lipids include, but are not limited to: ADC, 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOPTAC), 1 ,2-dioleoyl-3-(2- (dimethylamino)ethoxy)propylamine (DODEA), and 1 ,2-dimyristoyl-3- trimethylammonium-propane (DMTAP).

[0227] In some cases, an LNP includes an ionizable lipid. Examples of ionizable lipids include, but are not necessarily limited to: DLin-MC3-DMA, ALC-0315, SM-102, LP01 , CL1 , TCL053, CKK-E12, and analogs thereof. Other examples include: ATX-002, BP lipid 312, DLin-DMA, DLenDMA, DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin- K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLin-C2K-DMA, DLin-MP- DMA, DODMA, 98N12-5, C12-200, DLin-C-DAP, DLin-DAC, DLinDAP, DLinAP, DLin- EG-DMA, DLin-2-DMAP, KL10, KL22, KL25, LP01 , Lipid III-45, Octyl-CLinDMA, Octyl- CLinDMA (2R), and Octyl-CLinDMA (2S), and analogs thereof.

[0228] In some cases, an LNP includes a neutral phospholipid. Examples of neutral phospholipids include, but are not necessarily limited to: 5-heptadecylbenzene-1 ,3- diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1 ,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DM PE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE),lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0229] In some cases, an LNP includes a polymerizable lipid, e.g., a PEGylated lipid (e.g., DMG-PEG 2000, DSG-PEG 2000, ALC-0159). PEG-modified lipids (also referred to as PEGylated lipids) include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1 ,2-diacyloxypropan-3-amines. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG (DMG-PEG), PEG-DLPE, PEG DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid are 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG- DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG- distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1 , 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some cases, instead of being PEG- modified, the polymerizable lipid can be modified with other hydrophilic polymers (other than PEG) - e.g., in some cases, the lipids in this paragraph can be modified with a hydrophilic polymer other than PEG.

[0230] In some cases, an LNP includes a molecular payload such as nucleic acid and / or protein (e.g., a CRISPR-Cas effector protein, a DNA or RNA encoding a CRISPR-Cas effector protein, a guide RNA, a DNA encoding a guide RNA, or any combination thereof). LNP refers to a lipid-based vesicle useful for delivery of nucleic acid molecules and / or proteins and having dimensions on the nanoscale. In different embodiments the nanoparticle is from about 1 nm to about 1000nm, about 10nm to about 20nm, about 20nm to about 50nm, about 50nm to about 500 nm, or about 50nm to about 200nm.

[0231] Lipid nanoparticles may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g. “Iiposomes”-lamellar phase lipid bilayers that, in some embodiments are substantially spherical, and, in more particular embodiments can comprise an aqueous core, a dispersed phase in an emulsion, micelles or an internal phase in a suspension.

[0232] A variety of different nanoparticles can be employed, including LNPs, polymeric nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, e.g., Riley and Vermerris Nanomaterials 2017, 7, 94; Thomas et al., Molecules 2019, 24, 3744; Bochicchio et al., Pharmaceutics 2021 , 13, 198; Munagala et al., Cancer Letters 2021 , 505, 58; Fu et al., 2020 NanoImpact 20, 100261 ; and Neshat et al. 2020 Current Opin. Biotechnol. 66:1-10.).Kits

[0233] Provided are kits / systems for carrying out a subject method. Such kits comprise various combinations of components useful in any of the methods described elsewhere herein. In some embodiments a subject kit includes a nucleic acid encoding a subject CRISPR-Cas fusion protein and a nucleic acid encoding a deaminase fusion protein.

[0234] A kit can further include one or more additional reagents, where such additional reagents can be any convenient reagent. Components of a subject kit can be in separate containers; or can be combined in a single container. In some cases one or more of a kit’s components are pharmaceutically formulated for administration to a human.

[0235] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods (e.g., dosing instructions, instructions to administer the component(s) to an individual. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or subpackaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. Aswith the instructions, this means for obtaining the instructions is recorded on a suitable substrate.EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0236] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.1. A programmable base editor, comprising: a CRISPR-Cas fusion protein comprising a CRISPR-Cas effector protein fused to a first member of a split-protein binding pair, wherein the CRISPR-Cas effector protein is a nickase or is catalytically inactive, and a deaminase fusion protein comprising a deaminase protein fused to a second member of the split-protein binding pair, wherein the split-protein binding pair comprises a catcher / tag system or a GFP1-10 / GFP11 system.2. The programmable base editor of 1 , wherein one member of the splitprotein binding pair is a catcher protein, and the other member is a partner tag protein, and wherein the CRISPR-Cas fusion protein is covalently linked to the deaminase fusion protein via a post-translational isopeptide bond between the catcher protein and the partner tag protein.3. The programmable base editor of 2, wherein the catcher protein is SpyCatcherOOl , SpyCatcher AN 1 AC 1 , SpyLigase, SnoopCatcher, SpyCatcher002, SpyCatcher003, SnoopLigase, or SpyDock.4. The programmable base editor of 2, wherein the catcher protein comprises SEQ I D NO: 11.5. The programmable base editor of 2 or 3, wherein the partner tag protein is comprises SpyTag (AHIVMVDAYKPTK; SEQ ID NO: 1), KTag(ATHIKFSKRD; SEQ ID NO: 2), SnoopTag (KLGDIEFIKVNK; SEQ ID NO: 3), SpyTag002 (VPTIVMVDAYKRYK; SEQ ID NO: 4), SnoopTag Jr (KLGSIEFIKVNK; SEQ ID NO: 5), DogTag (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 6), or SpyTag003 (RGVPHIVMVDAYKRYK) (SEQ ID NO: 7).6. The programmable base editor of any one of 2-5, wherein the partner tag protein comprises RGVPHIVMVDAYKRYK (SEQ ID NO: 7).7. The programmable base editor of any one of 1-6, wherein the deaminase protein is an adenosine deaminase.8. The programmable base editor of any one of 1-6, wherein the deaminase protein is a cytidine deaminase.9. The programmable base editor of any one of 1-6, wherein the deaminase protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of any one of SEQ ID NOs: 32-35.10. The programmable base editor of any one of 1-6, wherein the deaminase protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 32.11 . The programmable base editor of any one of 1-6, wherein the deaminase protein is TadA-8e.12. The programmable base editor of any one of 1-11, wherein the CRISPR- Cas effector protein is an nCas9, dCas9, or dCas12a protein.13. The programmable base editor of any one of 1-11, wherein the CRISPR- Cas effector protein is an nCas9 protein.14. The programmable base editor of any one of 1-11, wherein the CRISPR- Cas effector protein is a dCas9 or dCas12a protein.15. The programmable base editor of any one of 1-14, wherein the first member of the split-protein binding pair is fused N-terminal to the CRISPR-Cas effector protein.16. The programmable base editor of any one of 1-15, wherein the second member of the split-protein binding pair is fused C-terminal to the deaminase protein.17. The programmable base editor of any one of 1-16, comprising one or more nuclear localization signals (NLSs).18. The programmable base editor of any one of 2-17, wherein the CRISPR- Cas effector protein is fused to the catcher protein, and the deaminase protein is fused to the partner tag protein.19. The programmable base editor of 18, wherein: the catcher protein is fused N-terminal to the CRISPR-Cas effector protein, the catcher protein comprises SEQ ID NO: 11, and the CRISPR-Cas effector protein is a nickase Cas9 protein (nCas9); and the partner tag protein is fused C-terminal to the deaminase protein, the partner tag protein comprises RGVPHIVMVDAYKRYK (SEQ ID NO: 7), and the deaminase protein is Tad-8e.20. A composition comprising the programmable base editor of any one of 1- 19, and a guide RNA.21 . A method of producing the programmable base editor of any one of 1-19, comprising contacting the CRISPR-Cas fusion protein with the deaminase fusion protein, thus producing the programmable base editor.22. The method of 21, wherein the split-protein binding pair comprises the catcher / tag system, wherein one member of the split-protein binding pair is a catcher protein, and the other member is a partner tag protein, and wherein said contacting results in covalent linkage of the CRISPR-Cas fusion protein with the deaminase fusion protein via post-translational isopeptide bond formation between the catcher protein and the partner tag protein.23. The method of 20 or 21 , wherein said contacting takes place at a pH of about 7.5.24. The method of any one of 21-23, wherein said contacting takes place at a salt concentration of about 300mM NaCI.25. The method of any one of 21-24, further comprising, prior to said contacting, producing the CRISPR-Cas fusion protein and the deaminase fusion protein.26. The method of 25, wherein said producing comprises expressing the CRISPR-Cas fusion protein and the deaminase fusion protein separately in cells, and extracting the CRISPR-Cas fusion protein and the deaminase fusion protein from the cells.27. The method of 25 or 26, wherein said producing comprises purification of the CRISPR-Cas fusion protein using affinity chromatography and gel filtration, and purification of the deaminase fusion protein using affinity chromatography.28. The method of any one of 25-27, wherein the CRISPR-Cas fusion protein and the deaminase fusion protein are purified at a pH of about 7.5.29. The method of any one of 21-28, further comprising, after said contacting, performing one or more purification steps to enrich for the programmable base editor.30. The method of 29, wherein said one or more purification steps comprise gel filtration.31 . The method of any one of 21-30, wherein the programmable base editor that is produced retains 80% or more deaminase activity when compared to the deaminase activity of the deaminase fusion protein prior to contact with the CRISPR-Cas fusion protein.32. A method for modifying a target nucleic acid, the method comprising: contacting a target nucleic acid with the programmable base editor of any of 1- 18, and a guide RNA, wherein the guide RNA hybridizes to a target sequence in the target nucleic acid, and wherein said contacting results in the deamination of an adenine or a cytosine of the target nucleic acid.33. The method of 32, wherein the target nucleic acid is genomic DNA.34. The method of any one 32 or 33, wherein the target sequence is a protein-coding sequence.35. The method of any one of 32-34, wherein said contacting takes place inside of a cell that comprises the target nucleic acid.36. The method of 35, wherein said contacting comprising introducing into the cell, (i) the programmable base editor, and (ii) the guide RNA or a nucleic acid encoding the guide RNA.37. The method of 35 or 36, wherein the cell is a eukaryotic cell.38. The method of 35 or 36, wherein the cell is a mammalian cell.39. The method of any of 35-38, wherein the cell is in vitro.40. The method of any of 35-38, wherein the cell is in vivo.41. The method of any of 36-40, wherein the programmable base editor and the guide RNA are introduced into the cell using a viral vector or a nanoparticle.42. The method of 41, wherein the nanoparticle is a lipid nanoparticle.43. A nucleic acid system comprising:(a) a first nucleotide sequence encoding the CRISPR-Cas fusion protein of any one of 1-19, or(b) a second nucleotide sequence encoding the deaminase fusion protein of any one of 1-19, or(c) both (a) and (b).44. The nucleic acid system of 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in a prokaryotic cell.45. The nucleic acid system of 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in an E. coli cell.46. The nucleic acid system of 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in a eukaryotic cell.47. The nucleic acid system of 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in a mammalian cell.48. The nucleic acid system of any one of 43-47, comprising a first nucleic acid comprising the first nucleotide sequence, and a second nucleic acid comprising the second nucleotide sequence.49. The nucleic acid system of 48, wherein the first and / or second nucleic acid is a plasmid.50. The nucleic acid system of 48, wherein the first and / or second nucleic acid is a viral vector.51 . The nucleic acid system of any one of 43-47, comprising a nucleic acid molecule that comprises both the first nucleotide sequence and the second nucleotide sequence.52. The nucleic acid system of 51 , wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters.53. The nucleic acid system of 51 or 52, wherein the nucleic acid molecule is a plasmid or a viral vector.54. The method of any one of 25 and 29-31 , wherein said producing comprises expressing the CRISPR-Cas fusion protein and the deaminase fusion protein in the same cell, thus leading to contact of the CRISPR-Cas fusion protein with the deaminase fusion protein and therefore formation of the programmable base editor in the same cell.55. The method of claim 54, wherein the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by and expressed from the same nucleic acid molecule.56. The method of claim 54, wherein the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by and expressed from the same plasmid.EXPERIMENTAL EXAMPLES

[0237] The following examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0238] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0239] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like.Example 1: Production of a programmable base editor

[0240] Programmable base editors (e.g., fusion of nCas9 with TadA-8e) have been challenging to produce with high yield via recombinant expression in E. coli, which limits its widespread use of base editor protein (and RNP) in gene editing applications. Cas9 nuclease protein is commercially available as a catalog productfrom many vendors, including as a version compliant with current good manufacturing processes (cGMP). In contrast, base editor protein is not commercially available for purchase, even as a research grade reagent.

[0241] Although genome editing is often performed using the protein-reliant RNP format when employing a nuclease, use of RNP has not been feasible for many base editing applications due to the challenge of producing high-yield, high-purity base editor protein. To deal with this bottleneck, the field often relies on delivery of mRNA encoding the base editor protein. For most CRISPR constructs, mRNA is more challenging to produce than recombinant protein, but for base editors the protein production challenge is such a substantial hurdle that mRNA is the best option available.

[0242] By using the CFIEP system disclosed herein to synthesize the base editor from its two constituent parts (each of which can be independently generated via recombinant expression / purification with high yield and purity), the provided compositions and methods increase the efficiency and yield of base editor production, making this type of protein a more appealing option for use in research and / or therapeutics.Materials and Methods

[0243] Expression and purification of TadA variants. E. coli BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding a TadA deaminase (TadA-8e) (see SEQ ID NO: 16). The resulting expression strains were grown overnight in Luria-Bertani (LB) broth supplemented with 20 piM ZnCh and 50 pg / mL of kanamycin at 37 °C. The cells were diluted 1 :100 into Terrific Broth (TB) and grown at 37 °C to OD6oo = ~1.0. The culture was cooled to 25 °C, and isopropyl -[3-d- 1- thiogalactopyranoside (IPTG) was added at 0.2 mM to induce protein expression. After ~5 h, the cells were collected by centrifugation at 4,000 g and resuspended in deaminase lysis buffer (20 mM HEPES [pH 7.5], 1 M NaCI, 10% [v / v] glycerol, 30 mM imidazole, 1 mM TCEP) with 1 mM phenylmethylsulfonyl fluoride. The cells were lysed by sonication (10 s pulse-on, 10 s pulse-off for 10 min total at 6 W output) and the lysate supernatant was isolated following centrifugation at 20,000 g for 30 min at 4 °C to remove cellular debris and recover the supernatant. The clarified lysate was loaded onto an equilibrated 5 mL HISTRAP™ FF column (Cytiva) prepacked with precharged nickel Sepharose to capture the His-tagged fusion protein. Subsequently, the column was washed with at least five column volumes of deaminase lysis buffer to remove non-specifically bound proteins. The His-tagged fusion protein was eluted using astep gradient of deaminase lysis buffer supplemented with 10-500 mM imidazole. TadA-containing fractions were pooled and concentrated by ultrafiltration (Amicon- Millipore, 10-kDa molecular weight cut-off) to ~1 ml_ total volume. The protein was diluted to 1 mg / mL in deaminase lysis buffer and quantified by SDS-PAGE to monitor protein purity and yield.

[0244] Expression and purification of nCas9 variants. E. coli Rosetta(DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding nCas9 (D10A in this instance) (see SEQ ID NO: 24). The resulting expression strains were grown overnight in Luria-Bertani (LB) broth containing 100 pg / mL of ampicillin and 34 pg / mL chloramphenicol at 37 °C. The cells were diluted 1:100 into Terrific Broth (TB) and grown at 37 °C to ODeoo = ~1.0. The culture was cooled to 18 °C, and isopropyl - P-d-1- thiogalactopyranoside (IPTG) was added at 0.2 mM to induce protein expression. After ~20 h, the cells were collected by centrifugation at 4,000 g and resuspended in nickase lysis buffer (20 mM HEPES [pH 7.5], 1 M NaCI, 10% [v / v] glycerol, 10 mM imidazole, 1 mM TCEP) with 1 mM phenylmethylsulfonyl fluoride. The cells were lysed by sonication (10 s pulse-on, 10 s pulse-off for 10 min total at 6 W output) and the lysate supernatant was isolated following centrifugation at 20,000 g for 30 min at 4 °C to remove cellular debris and recover the supernatant. The clarified lysate was loaded onto an equilibrated 5 mL HISTRAP™ FF column (Cytiva) prepacked with precharged nickel Sepharose to capture the His-tagged fusion protein. Subsequently, the column was washed with at least five column volumes of nickase lysis buffer to remove non-specifically bound proteins. The His- tagged protein eluted in nickase lysis buffer supplemented with 300 mM imidazole. The eluted sample was next loaded onto a 5 mL HiTrap Heparin HP column (Cytiva) equilibrated in 20 mM HEPES (pH 7.5), 300 mM NaCI, and 10% (v / v) glycerol. The column was washed with at least five column volumes of equilibration buffer to remove non-specifically bound proteins before being loaded onto a HiLoad 16 / 60 S200 Superdex column (Cytiva) using an AKTA system. The protein was eluted with a linear gradient of 0.3-1 M NaCI in 20 mM HEPES and 10% (v / v) glycerol buffer (pH 7.5) containing 1 mM TCEP. Nickase-containing fractions were pooled and concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cut-off) and quantified by SDS-PAGE to monitor protein purity and yield.

[0245] Conjugation of TadA and nCas9. Protein conjugation was performed by incubating equimolar amounts of deaminase and nickase proteins from above (see SEQ ID NOs: 16 and 24) in conjugation lysis buffer (20 mM HEPES [pH 7.5], 300 mM NaCI, 10%[v / v] glycerol) at a final protein concentration of 1 mg / mL. The reaction mixture was incubated at room temperature for 1 hour with gentle agitation. Following incubation, the conjugated protein sample was concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cut-off) and loaded onto a HiLoad 16 / 60 S200 Superdex column (Cytiva) using an AKTA system equilibrated with gel filtration buffer (20 mM HEPES [pH 7.5], 300 mM NaCI, 10% (v / v) glycerol). Protein was loaded in volumes no greater than 2 ml_. Fractions containing the conjugated protein complex were pooled and concentrated for further analysis (Amicon-Millipore, 100-kDa molecular weight cut-off). Purified proteins were concentrated to ~50 pM, filtered using Spin-X centrifuge tube (0.22 pM, Corning) and stored at -80 °C.Results

[0246] FIG. 2 SpyTag / Spycatcher was conjugated to TadA-8e and nCas9 (referred to herein as base editor domains) to form a programmable base editor. Lanes 1 and 2: constituent domains of base editor on SDS-PAGE; each domain was fused to a SpyTag or SpyCatcher, making each compatible with SpyTag / SpyCatcher conjugation. Lanes 3 and 4: after mixing base editor domains together (Cas9 nickase and TadA-8e deaminase), a band corresponding to base editor MW appeared, demonstrating proof-of-concept conjugation (3) (Without fusion of the TadA and nCas9 to the catcher protein / partner tag protein (e.g., SpyCatcher / SpyTag, no conjugation would be observed); subsequent purification steps removed unwanted byproducts to achieve high purify base editor (4).Example 2: Editing with the engineered programmable base editor

[0247] After the BE (base editor) components were conjugated (see example 1), activity of the editor was validated in terms of DNA editing. B2M, a very common targeting loci for editing validation studies, was targeted. Importantly, beyond testing activity, a direct editing comparison to a commercially supplied ABE was performed. The ABE was specially ordered from a contract research organization (CRO). The results were very exciting because the demonstrate not only that the new base editors achieve similar or superior editing results vs. commercial ABEs, but also that the conjugation strategy allows for much larger protein yields (up to 10x) than conventional production strategies. This will provide a major upgrade for both research and industry applications.

[0248] FIG. 3 Percent editing efficiencies of CFIEP-made and commercial ABE B2M- knockout via PERC in primary human T cells, (left) Adenine base editor (ABE) B2M- RNPs engineered by conjugation following independent expression and purification (CFIEP), compared to a commercially available ABE supplied by a contract research organization (CRO). The ABEs were delivered via peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC; see, e.g., Foss et al., Nat Biomed Eng. 2023;7(5):647-660). Knockout efficiencies were assayed 4 days after delivery by flow cytometry. 50 pmol of RNP were delivered, (middle) ABE edited cell yield measured by total number of B2M-knockout cells, (right) Primary human T cells were stained with live-dead stain and surface marker-targeting antibodies and sampled at defined volumes to quantify cell counts; NT = no treatment.Example 3: Comparison of protein yields from recombinantly expressed adenine base editor

[0249] FIG. 5. Adenine base editors (ABEs) were expressed and purified by four different sources. Commercially available ABE was supplied by a contract research organization (CRO). The QB3 MacroLab and Wilson Lab (WL) also prepared ABEs by single-plasmid expression and 3-step purification in contrast to ABEs engineered by conjugation following independent expression and purification (CFIEP); n=3.Example 4: Relative editing efficiencies mediated by commercially- or CFIEP- produced cytosine base editor in primary human T cells

[0250] FIG. 6. Primary human T cells were edited using 100 pmol of cytosine base editor (CBE) RNP targeting the T cell receptor beta chain (TRBC) delivered via A5K peptide (10 uM). Three days after editing, cells were stained and analyzed using flow cytometry for T cell receptor (TCR) knockout. Comparison of a commercially purchased CBE shows comparable knockout rates with the CBE engineered by conjugation following independent expression and purification (CFIEP). In this illustrative example, the deaminase fusion protein included cytosine deaminase fused to SpyTag [TadA-8e-CD, see SEQ ID NO: 18 for the sequence plus the SpyTag and SEQ ID NO: 19 for the sequence without the SpyTag, also see FIG. 4], while the CRISPR-Cas fusion protein included nCas9 fused to SpyCatcher. Because CBEs also in some cases include uracil glycosylase inhibitor (UGI) domains, in this example the nCas9 was fused to SpyCatcher and to UGI [SpyCatcher-nCas9-UGI, see SEQ ID NO: 73, also see FIG. 4],Example 5: High yield production of DNA base editing ribonucleoproteins via CRISPR-Cas and deaminase fusion: Versatile base editor enzyme production improves purity, yield, and activity

[0251] This example includes some information from the above examples, but also includes additional information.

[0252] Base editors (BEs) are transformative genome editing tools that enable precise single-base conversions without reliance on double-stranded breaks. However, production of high-yield and high-purity BE protein is challenging, limiting the use of BEs in ribonucleoprotein (RNP) format in research and therapeutic settings. The work herein reports a modular approach for assembling high-yield, high-purity adenine and cytosine BEs via separated translation events and post-purification conjugation of the two constituent domains: a CRISPR-Cas effector protein (e.g., CRISPR-Cas9) and a deaminase. This system, dubbed BEaST (base editor assembled via separated translation), leverages the SpyTag / SpyCatcher system to covalently link deaminase and nickase polypeptides following their recombinant expression in bacteria. The BEaST approach can provide ~10-fold higher protein yields as compared to the traditional expression of a BE fusion and the resulting high-purity protein can be used to produce fully active BE RNPs. BEaST RNPs have been evaluated (herein) in multiple cellular contexts, including cultured primary human T cells and hematopoietic stem cells as well as murine neurons in vivo. BEaST RNPs mediate genome editing efficiency that often surpasses that of traditionally-produced BE fusion proteins, and the modular nature of BEaST facilitates streamlined production of Cas9 variants and / or deaminase variants, facilitating screening efforts. The BEaST platform addresses a longstanding manufacturing challenge, allowing convenient use of potent base editor enzymes ex vivo and in vivo.

[0253] Genome editing technologies have revolutionized the ability to understand and manipulate genes. These technologies have vast potential for applications in both basic science research and applied therapeutics. Base editing is a broadly useful example of a CRISPR-based genome editing approach that has rapidly been adopted in the laboratory as well as the clinic. Programmable base editors (BEs) have emerged as powerful tools for precision genome engineering, typically enabling targeted C«G-to-T«A or A«T-to-G«C conversions without reliance on double-stranded DNA breaks or homology-directed repair. BEs traditionally consist of a genetic fusion between an attenuated nickase Cas9 (nCas9) domain that mediates genomictargeting and a deaminase domain that acts on either adenine or cytosine, ultimately resulting in a genetic change to guanine or thymine, performed by ABE or CBE enzymes respectively. BEs have been widely applied across mammalian systems and hold therapeutic potential for a range of genetic diseases. However, despite widespread utility, the production of functional, high-purity BE ribonucleoproteins (RNPs) at scale is a substantial limitation for research and clinical use.

[0254] BE effectors can be delivered in several cargo formats: DNA encoding the BE protein as well as the guide RNA (gRNA) (typically as plasmid or via a viral vector); mRNA encoding BE protein, co-delivered with gRNA (typically within a lipid nanoparticle or via electroporation); as a pre-formed ribonucleoprotein (RNP) enzyme comprising protein and gRNA (typically via electroporation, virus-like particle, lipid nanoparticle, or amphiphilic peptides). RNP-format delivery is particularly appealing and is the default approach used by essentially all ex vivo therapeutic programs employing CRISPR nuclease (including Casgevy, the first FDA-approved CRISPR therapy). Delivery of RNP is advantageous because it introduces a brief pulse of active CRISPR effector, mediating efficient editing while minimizing off-target editing as compared to mRNA delivery. However, despite the appeal of the RNP format, commercial production of the required BE protein via recombinant expression in E. coli at the yield and purity required for therapeutic applications has remained unattainable, and consequently the field has relied on DNA- or mRNA-format delivery of BEs instead. Indeed, clinical use of BE ex vivo and in vivo has been limited to mRNA format to date. The impracticality of generating BE protein has been a firm impediment to the clinical use of BE RNP, exposing clinical trial subjects to elevated risks of off-target editing. This issue extends to the dearth of commercially available BE protein for research use, which is in stark contrast to the Cas9 nuclease sold by multiple vendors, including in the form of a GMP-grade product suitable for clinical use. Academic efforts have been able to produce enough BE protein to enable encouraging pre-clinical efforts using BE RNPs, but there has been an insurmountable impediment that precludes clinical use of BE as RNP: the challenges of BE protein production.

[0255] Traditional BE production involves co-expression of deaminase and nCas9 as a single polypeptide in E. coli, often resulting in poor solubility, compromised purity, and low yields. Attempts to increase expression or purification efficiency have included modifications to codon usage, solubility-enhancing tags, and chromatographic strategies, with limited success. Additionally, batch-to-batch variability and the complexity of expressing multi-domain proteins hinder scalability and quality control. Itis unclear why high-yield production of traditional BE protein has been such a substantial challenge to date; it may be due to the relatively large size of the fusion construct (-200 kDa) or due to differing optimal expression conditions for the constituent components (deaminase and nickase). The status quo for BE protein production involves recombinant expression in E. coli followed by purification via a series of chromatography steps. Expression of an intact BE construct is unpredictable and seldom generates sufficient protein for most use cases. This is further complicated by frequent challenges associated with purifying the intact BE construct. A final challenge is that the resulting BE protein sample is often heavily contaminated with other proteins and / or BE protein fragments, making it challenging to quantify the functional BE in the heterogeneous solution. Ideally, production would result cytosine base editors (CBEs) or adenine base editors (ABEs) with >95% purity and -10 mg per liter of E. coli cell culture, as is possible when producing Cas9 nuclease. However, even optimized protocols for ABE purification from human cells still only yield 1-2 mg of purified protein per liter of suspension cell culture and protein purity is often poor.

[0256] To address these challenges, a modular platform was developed for BE protein production that relies on independent translation of the two constituent domains followed by conjugation.

[0257] BEaST (Base Editor assembled via Separated Translation) is a method that exploits the covalent SpyTag I SpyCatcher interaction, to fuse a deaminase domain to a CRISPR-Cas effector protein (e.g., Cas9). This approach facilitates the independent optimization and purification of each component, followed by ligation (e.g., controlled ligation) to form a functional BE RNP. BEaST facilitated high purity production of a dozen distinct CBE and ABE enzymes, with yield improvements as high as -10-fold over the traditional fusion approach. BEaST ABE RNPs were fully active in cultured primary human T cells and hematopoietic stem cells, also supporting in vivo gene editing in murine neurons when delivered to the brain parenchyma via convection- enhanced delivery (CED). BEaST production facilitated the direct comparison of several BE configurations, including distinct PAM variants, the number of active deamination domains, deamination window variants, and different catalytic states of Cas9 (nickase vs. nuclease-deficient). The BEaST strategy provides a scalable, flexible platform for production and evaluation of ABE and CBE enzymes in RNP format, lowering barriers for research and clinical base editing with potent on-target editing and minimized off-target effects.ResultsHigh-yield and high-purity production of base editors via BEaST

[0258] To circumvent the limitations of established single-plasmid expression methods, the BEaST workflow was developed, which decouples the production of a given BE’s deaminase and nCas9 components (Fig. 7). The deaminase domain, initially a catalytically active TadA-8e fused to a catalytically inactive dTadA-8e (as previously characterized), was expressed bearing a C-terminal SpyTag. Separately, nCas9 was expressed bearing an N-terminal SpyCatcher domain. Both proteins were expressed in E. coli, partially purified in parallel via nickel and heparin affinity columns, and then covalently ligated via SpyTag / SpyCatcher to yield an intact BE RNP. The resulting BEaST protein is further purified via size exclusion chromatography (SEC), removing nucleic acid contaminants and any unreacted deaminase. The conjugation step employed an intentional excess of deaminase (over nCas9), which helps ensure that no free nCas9 remains. Free nCas9 could act as a competitive inhibitor of the BE in cells, and it is not readily separated from intact ABE via SEC, hence the importance of conjugating as much nickase as possible. The resulting BEaST ABE resembled a traditional ABE (tABE) produced as a single fusion construct, with the major distinction being the presence of a 14 kDa SpyTag I SpyCatcher domain protruding from the middle of the linker region (typically 32 amino acids for tABE).

[0259] Compared to tABE from a commercial source and from an academic protein production core, BEaST ABE had higher purity (Fig. 7b) and produced ~10-fold higher protein yield per liter of bacterial culture (Fig. 7c). This production advantage was consistent across multiple independent preparations, where BEaST ABE reproducibly outperformed tABE in both yield and purity.Functional comparison of BEaST and traditional ABE RNPs

[0260] Activity of BEaST ABE and CBE RNPs were evaluated in primary human T cells by delivering the RNPs using either electroporation or peptide-enabled RNP-format CRISPR (PERC) delivery (Fig. 8a). A splice-mediated strategy was used for knockout of B2M, an edit that has been used to enhance cell therapies in the context of allogeneic transplant. In a pilot experiment, a BEaST enzyme bearing a single active TadA-8e domain (BEaST-1 *) was compared to a CRO-produced traditional ABE (tABE) with a similar domain architecture, and both enzymes mediated efficient B2M knockout (Fig. 8b). Based on this encouraging initial result, several more BEaST enzymes were generated to assess their activity and evaluate the capacity for thenew approach to facilitate combinatorial production of a diverse enzyme array (Fig. 8c) using just a few domains as starting materials.BEaST-mediated editing in primary human T cells using tandem active TadA domains

[0261] Prior structural studies have shown that TadA deaminase domains, when expressed as monomers, can have a natural tendency to form intermolecular dimers. This dimerization behavior can lead to the formation of multimeric BE RNPs, which may hinder genomic search efficiency and increase the risk of aggregation. To circumvent these potential liabilities, all tABE and BEaST proteins in this study feature a preformed dimeric deaminase unit, where two TadA domains are connected by a flexible linker to the N-terminus of Cas9. This domain architecture is intended to promote intramolecular TadA dimer formation, to the exclusion of intermolecular dimerization.

[0262] The BEaST construct initially evaluated incorporated a single active TadA domain fused to a catalytically deactivated TadA (dTadA) partner, a configuration referred to here as “1x”. Because BEaST enables rapid production of different BE variants, a construct containing two catalytically active TadA domains was next generated, termed “2x”, designed to potentially double the deamination capacity of the editor thereby improving editing potency. Direct comparison of these constructs revealed that the BEaST-2x configuration out-performed BEaST-1 x when delivered via PERC (Fig. 8e). A construct that bearing an additional two additional active TadA domains at the C-terminal end of nCas9, dubbed BEaST-4x, was generated to test the hypothesis that more domains can further enhance activity. The BEaST-4x construct had only marginal (and not statistically significant) improvements in activity overall base editing efficiency for B2M knockout (Fig. 8e, top), suggesting that the BEaST-2x may provide sufficient deaminase density to reach maximal editing efficiency for a given scenario.

[0263] In parallel, BEaST constructs with different PAM-recognition properties were also evaluated. An inherent strength of base editing is its precision: only a 6-8 nucleotide window is typically targeted. But the downside of this precision is that given target locus requires a proximal PAM compatible with the CRISPR domain being used: typically, the NGG of SpCas9. Many efforts have dealt with this challenge by employing Cas9 domains featuring altered PAM specificity, either stemming from natural variation, rational design, or by continuous evolution. A recent flurry of effort has produced scores of novel Cas9 variants with diverse PAM recognition properties,and one of these enzymes (featuring an NGC PAM) was employed in the rapid production of individualized ABE therapy for a single patient. The capacity for BEaST to produce PAM variant ABE RNPs was assessed by replacing the typical SpCas9 nCas9 domain (NGG PAM) with the SpCas9-NG variant (NG PAM) (Fig. 8c). Although use of a relaxed PAM variant can result in lower activity (likely due to the altered kinetics of the enzyme’s target site search), the BEaST format promoted activity comparable to that of the analogous tABE enzyme (Fig. 8d,e).

[0264] To evaluate how the orientation of deaminase relative to Cas9 affects activity, TadA- 8e constructs were tested with SpyTag fused to either the N- or C-terminus. C- terminal SpyTag positioning led to higher editing activity in primary T cells, suggesting improved accessibility or orientation of the deaminase when fused at this site. Accordingly, although the N-terminal fusion functioned successfully, the BEaST deaminases feature a C-terminal SpyTag fusion throughout the work described in this example.BEaST supports fine-tuning of the base editing activity window

[0265] Base editing is mediated by action of a deaminase on nucleobases that are exposed on the non-target DNA strand following complexation between the gRNA’s spacer sequence and the target DNA strand (a structure known as an R-loop). For a given BE, there is a characteristic window of efficient deamination activity, typically spanning nucleotides 4-9 when counting from the 5' end of the gRNA, with peak activity at (or near) position 6. Depending on the specific properties of the target locus and the intended edit, the enzyme with a narrower deamination window may be preferable. Bystander editing refers to the deamination of a nucleobase adjacent to the intended target nucleobase (typically at one edge of the BE window), an editing outcome that may have deleterious consequences in some therapeutic use cases. This has led to development of deaminase variants with distinct preferences, including variants exhibiting a narrowed BE window. Facilitated by the modular approach to protein production discussed herein, BE window properties were readily compared using a BEaST-format ABE RNP incorporating two distinct engineered TadA-8e variants. The N108Q, L145T variant of TadA (dubbed “QT” here) has been previously reported to reduce bystander activity. The TTI variant (S7T / A114T / F148I) was recently developed using directed evolution in E. coli.

[0266] Echoing the results with the NG PAM ABE, the activity of these narrow-window ABEs tends to be diminished compared to standard ABEs (Fig. 8d,e); there is an apparenttrade-off between activity and specificity. This decrement tends to be more apparent when using transient delivery of RNP, a therapeutically-relevant approach that avoids the persisting expression that is typically employed during the development of novel enzyme variants (masking deficiencies in absolute activity by saturating the cell with effector).BEaST enzymes can be used largely interchangeably with traditional ABE enzymes

[0267] Because the relative orientation of the deaminase and Cas9 domains can influence the nature of the deamination window - and the BEaST linker differs slightly from the tABE linker - two gRNAs were evaluated to assess any impacts on the resulting BE activity window (Fig. 9). The window was evaluated using two gRNAs targeting a therapeutically-relevant locus capable of elevating fetal hemoglobin (Fig. 9b) and we observed a slight change in the propensity for A-to-G editing across the window, with tABE slightly favoring the 5' side of the window and BEaST slightly favoring the 3' side of the window. These differences in activity may be a consequence of the distinct linker properties. It is expected that for most use cases the distinct BE window will not complicate the use of BEaST interchangeably with tABE. For some therapeutic target loci, the distinct activity window of BEaST may be advantageous: a locus could feature a deleterious bystander nucleotide near the 5' end of the window, and in that case, BEaST may be preferable to tABE.

[0268] By normalizing for the total base editing at the candidate nucleotides in the BE window for a given gRNA, the fractional editing efficiency of each adenine were compared, thereby conveying the general character of the BE activity window for the distinct enzymes (Fig. 9c, right). The activity window was largely comparable for BEaST enzymes and tABE enzymes, suggesting BEaST can be used interchangeably with tABE enzymes in most cases. Unexpectedly, the QT variant did not produce markedly narrowed window activity as compared to the analogous BEaST enzymes bearing a standard TadA-8e domain (Fig. 9c, right). This may be a locus-specific result, and it is possible that editing at other loci would produce the anticipated narrowed window of activity. Indeed, the TTI variant showed striking sensitivity to the locus being targeted: shifting the guide / target by just one nucleotide altered the enzyme’s window from comparable to the analogous BEaST enzymes (the “AGG” gRNA) to markedly narrowed (the “GGG” gRNA) (Fig. 9c, right). At the latter locus, the TTI enzyme (with either NG or NGG PAM compatibility) showed a strongpreference for position A7, with -80% of all editing at that position and <10% editing at the A4 and A9 positions (Fig. 9c, right). This is in contrast to the non-TTI enzymes, where -55-60% of the editing occurred at the A7 position, with -20% editing at the A4 and A9 positions. This is a narrow activity window and may have utility in therapeutic applications where bystander editing would be detrimental.In vivo brain editing using BEaST ABE

[0269] Based on previous reports of NLS-rich Cas9 performing self-delivery into neurons when RNPs were administered into the murine striatum using convection-enhanced delivery (CED), we were curious whether BEaST ABE RNP would exhibit similar activity by virtue of the four NLS motifs the protein bears. The GER10 reporter mouse was employed, which harbors a green fluorescent protein (GFP; mVenus) cassette that is repressed until ABE activity eliminates a premature stop codon. Following CED administration of BEaST ABE RNP into the GER10 mouse striatum, robust genome GFP signal was observed that approached 100% base editing efficiency near the site of injection (Fig. 10). The coverage and editing efficiency - 17% of the striatum exhibiting evidence of editing with 38% neuronal editing efficiency within that area - suggest that BEaST RNPs can edit with high efficiency but may not spread throughout the brain as well as NLS-rich Cas9 does. Nevertheless, BEaST ABE RNPs exhibited robust activity in vivo, following carrier-free administration. It is expected that BEaST enzymes will be fully compatible with the diverse technologies that have been used to promote RNP delivery in various organs and tissues, establishing therapeutic candidates targeting the brain, eye, lung, ear, or immune cells.

[0270] Whole-section imaging of GER10 mice after CED of buffer or ABE revealed a discrete GFP-positive domain confined to the right dorsal striatum at the injection site (arrows), with negligible contralateral signal (scale bar, 1 mm). High-magnification confocal laser-scanning microscopy (CLSM, 20x) confirmed the absence of GFP in the control striatum (top row) and dense GFP labeling within the targeted striatum (bottom row). NeuroTrace staining delineated neuronal somata, and merged images showed robust co-localization of GFP and neurotrace (scale bar, 50 pm). ROI-based analysis yielded two prespecified metrics (Fig. 10c, d): percent striatum edited, the fraction of striatal tissue covered by the GFP distribution map (16.75%), and percent neuronal editing within the edited region, the fraction of NeuroTrace-positive cells whose DAPI- segmented nuclei met the >50% GFP-core overlap criterion (38.4%). The bar plots(mean with individual sections overlaid) recapitulate these findings, indicating substantial striatal coverage with a high proportion of GFP-positive neurons and minimal signal contralaterally, consistent with efficient, local ABE delivery following CED.Optimization further improves BEaST yield, purity, and activity

[0271] While the majority of the experiments described in this study were performed using the original BEaST protocol, further optimization substantially improved production outcomes. Specifically, TadA expression was refined by increasing the IPTG concentration from 0.2 mM to 0.5 mM and introduced a heparin purification step prior to conjugation. These changes led to a significant increase in soluble TadA yield. The addition of the heparin chromatography step also markedly decreased nucleic acid contamination - an issue observed in earlier preparations - further improving the purity of the resulting BEaST RNPs. Conjugation with nCas9 proceeded more efficiently under these updated conditions, yielding BEaST RNPs with improved biochemical homogeneity and enhanced in vitro editing activity.Discussion

[0272] Genome editing technologies have ushered in a new era of precision manipulation of DNA, offering unparalleled potential for scientific research and therapeutic applications. Among these tools, BEs have emerged as powerful and versatile effectors with immense utility in research and therapeutic applications. However, BE protein production and purification has remained a major impediment for the use of pre-formed RNP complexes in genetic research and clinical genome editing. Most delivery of BE has relied on the use of mRNA, a format that mediates efficient editing but is associated with substantially increases rates of off-target editing as compared to base editing mediated by pre-formed RNP enzymes. BE-mediated off-target editing takes place at genomic loci resembling the target site, genomic loci unrelated to the target site, and in RNA transcripts. The latter two classes of unintended editing can be mediated by a deaminase domain that is not associated with gRNA, a species that is likely prominent in the case of mRNA-format delivery. RNP format likely results in less surplus deaminase within the cell, and thus lower rates of unintended deamination. Based of its robust activity and well-established safety advantages, we contend that RNP format BE delivery would be much more widespread - especially in therapeutic contexts - if BE protein were readily available with high yield and purity.

[0273] To address this need, BEaST was developed, an approach to base editor production that separates the protein into two separate polypeptides that are independently translated and subsequently assembled using a two-piece self-assembling domain featuring a covalent bond (the SpyTag / SpyCatcher pair). This approach was taken by realizing that genetically fused BE proteins are challenging to produce in bacterial hosts due to the large size and multi-domain structure of these chimeric proteins. BEaST editors represent a significant advance in producing a diversity of BE enzymes paving the way for broader applications. BEaST-mediated BE protein production provides an alternative to genetically-fused recombinant BE protein, which often suffers from low yields and inadequate purity. The BEaST strategy increases the yield and purity of optimally active BE production, facilitating widespread use of BE in preformed RNP format, which has substantial value for both research and therapeutics. The plug & play nature of this approach has facilitated combinatorial production and evaluation of a variety of novel BE enzymes that would have otherwise been intractable to produce. Any research team dedicated to development of base editing strategies can readily produce BEaST-compatible components - an array of deaminases and an array of CRISPR domains - to allow facile pairings and a multiplicative wealth of resulting BE enzymes. For example, purification of four TadA variants and six nCas9 variants would provide rapid access to up to 24 diverse BE enzymes. Indeed, we are unaware of any prior study that has compared as many BE enzymes in RNP format as the 15 we have here, a testament to the value in the BEaST approach.

[0274] The BEaST platform addresses key production and functional bottlenecks in the field of base editing. By decoupling expression of deaminase and Cas9 components and using covalent protein ligation post-purification, BEaST enables high-yield, high-purity production of active BE RNPs. This stands in contrast to prior approaches requiring co-expression of multi-domain fusion proteins, which often suffer from low solubility and heterogeneous products. This work complements and extends previous efforts in improving BE production by introducing a flexible assembly platform that separates expression from final functional assembly. Functionally, BEaST RNPs perform equivalently or better than commercially sourced or conventionally expressed BEs. The modular nature of BEaST allows for streamlined testing of multiple protein configurations, as well as facile switching between BE classes. Moreover, compatibility with multiple delivery modalities - electroporation, PERC, and CED - suggests broad utility in both ex vivo and in vivo contexts. Notably, successful in vivoediting in the murine striatum highlights the potential for BEaST editors to be further engineered for enhanced self-delivery.

[0275] A strength of the BEaST platform is its modular architecture, which allows iterative optimization of each component without altering the overall assembly strategy. Toward the end of this study, a refined protocol for TadA expression was implemented that significantly improved the production pipeline. This updated approach included increasing the IPTG concentration during expression (from 0.2 mM to 0.5 mM) and adding a heparin affinity chromatography step prior to conjugation. These adjustments not only improved soluble yield and conjugation efficiency but also reduced nucleic acid contamination that had been present in earlier TadA preparations. Though most data presented here were generated using the original protocol, this late-stage enhancement yielded BEaST RNPs with superior purity and higher in vitro editing performance. Together, these findings underscore BEaST’s adaptability and translational potential. Improvements to individual modules can propagate downstream benefits without requiring a complete redesign, strengthening the platform’s long-term utility for scalable, high-quality BE production.

[0276] The BEaST platform facilitates generation of a library of purified Cas9 and TadA protein components that can be rapid and efficient assembled to create bespoke BE enzymes. A recent CRISPR success story relied on use of an ABE featuring an evolved nCas9 with an “NGC” PAM preference. This nCas9 variant could readily be on-boarded. By independently purifying and storing numerous variants of each protein, researchers can establish a versatile toolkit analogous to a chemical reagent library. This strategy allowed us to quickly test unconventional TadA variants like BEaST-4x, which was variable in its capacity to surpass the activity of the other enzymes depending on the locus tested, possibly due to steric interference or complex interactions with the R-loop formed at a given target locus. Nonetheless, when a specific BE is required, simply retrieving the corresponding Cas9 and TadA proteins from storage, combining them, and performing size-exclusion chromatography (SEC) yields a functional BE within a matter of hours. This approach significantly streamlines the BE production process, reducing the turnaround time from weeks to days.

[0277] Given the increasing adoption of CRISPR-based therapies for clinical and research applications, the format in which editing components are delivered has become an important determinant of both efficacy and safety - whether as mRNA or as protein in the form of RNPs - significantly influencing editing outcomes. mRNA is easilydegraded and has low stability. In particular, gene-editing tools that deliver Cas9 to function in concert with effector proteins are difficult to apply because the number of bases in the mRNA encoding Cas9 and effector proteins is too large. Compared to DNA and mRNA formats, RNPs are considered safer. Studies have shown that RNP delivery tends to result in higher on-target editing efficiency and lower off-target mutations compared to other methods. Additionally, delivering Cas9 as a purified protein allows for immediate nuclear gene editing and, therefore, has higher efficiency than DNA- or mRNA-based methods. The BEaST platform makes RNP delivery of BEs a practical and scalable option.

[0278] The findings here show that ABE8e delivered as an RNP complex achieves editing efficiencies comparable to mRNA delivery at the early time point (Day 3 postelectroporation), with efficiency continuing to increase over time. Notably, RNP delivery offers the added advantage of reducing gRNA-dependent off-target editing relative to mRNA as observed in similar studies, likely due to the shorter intracellular exposure time of the editor. In parallel, the DNA break-free variant dABE8e provides a safer editing approach that further improves cell recovery. Because editing outcomes are dose-dependent, systematic titration of mRNA or protein with sgRNA will be necessary to define the optimal conditions that maximize on-target efficiency while minimizing off-target activity and maintaining high cell viability, an essential step toward clinical application.

[0279] This endeavor marks a critical step forward in harnessing the full potential of BE RNPs, ultimately unlocking new frontiers in genome manipulation for research and therapeutic applications. The capacity for the commercially available Cas9 nuclease protein to be produced at high yields allowed Cas9 RNP to be used in the clinic and is now the basis of CRISPR drugs that have been approved for use in hemoglobinopathies. In contrast, BEs have not been widely used as RNP, almost certainly due to the extreme difficulty and unpredictability of BE protein production and as a result are not commercially available for purchase, even as a research grade reagent. Research and biopharma efforts instead often rely on mRNA for BE delivery, a cumbersome solution that is nevertheless more appealing than trying to make high yield BE protein. The approach here could improve access to BE protein, allowing base editing via delivery of RNP, a more appealing format than mRNA (which is technically challenging and can have negative impacts on cells). By using the BEaST system to synthesize the BE from its two constituent parts, this protein production pipeline increases the efficiency, quality, and yield of BE proteins, making the cargoclass a more appealing option for use in research and / or therapeutics. With its yield, purity, and modularity, BEaST offers a path forward for producing scalable and clinically relevant genome editing reagents. As therapeutic genome editing progresses toward clinical translation, BEaST provides a robust and tunable strategy for producing the next generation of precision genome editors.Methods Cell Culture

[0280] Isolated T cells were thawed (on day -3 relative to delivery) and cultured overnight in X-VIVO 15 medium (Lonza) supplemented with 5% fetal bovine serum (FBS), 50 pM 2-mercaptoethanol and 10 mM A / -acetyl-L-cysteine. Cells were activated (on day -2 relative to delivery) and cultured at 1 x 106cells mL"1for 2 days in supplemented X- VIVO 15 medium with anti-human CD3 / CD28 magnetic Dynabeads (Gibco 40203D) at a bead-to-cell ratio of 1 :1 , 200 U mL-1IL-2 (Proleukin), 5 ng mL-1IL-7 (R&D Systems) and 5 ng mL-1IL-15 (R&D Systems). After 2 days of activation, Dynabeads were removed from the cell culture using an EasySep cell separation magnet (STEM CELL). For genome editing, 200 x 1O3T cells per well were suspended in 100 pl Opti-MEM (Gibco) (treatments detailed below). After 1 h of treatment, FBS- supplemented X-VIVO-15 recovery medium was added to cells. Unless specified otherwise, after genome editing (as described below), cells were cultured in supplemented X-VIVO 15 at 0.5 106cells mL-1with 300 U mL'1IL-2 (Proleukin) and split every 2-3 days. Cell viability was assessed using a CellTiter-Glo assay (Promega G7570) according to the manufacturer-provided instructions. Luminescence was measured using a Spark plate reader.Peptides

[0281] TAT peptide was purchased from GenScript (GSCRPT-RP20256), and other peptides were procured via custom solid phase synthesis (CPC Scientific; 95% purity). All peptides were stored lyophilized or as 10 mM stocks in DMSO at -20 °C in a desiccator.Cloning

[0282] All cloning was conducted by Gibson Assembly Master Mix enzyme cloning methods (New England Biolabs). DNA templates were derived by PCR amplification and carried out using Q5 High Fidelity DNA Polymerase (New England Biolabs). Allprimers and gBIocks Gene Fragments used in this work were obtained from Integrated DNA Technologies. Vectors were transformed into XL-Blue competent cells (Agilent Technologies) prepared by UC Berkeley MacroLab. All plasmids used in this work were freshly prepared from 5 mL of XL-Blue cell culture using QIAprep Spin Plasmid Miniprep (QIAGEN). Molecular biology grade, DEPC-treated water was used in all assays, transfections, and PCR reactions to ensure exclusion of DNAse activity.TadA variant TTI by directed evolution in E. coli

[0283] Targeted mutagenesis on the TadA domain was performed with Mutazyme II (AGILENT) following the manufacturer’s instructions, targeting a medium mutation rate (-5-10 mutations per kilobase). The mutagenesis library was assembled via golden gate assembly using PaqCI (NEB) into a vector containing dCas9 and sgRNA cassette driven by T7 promoters (p15A, CamR). The assembled library was transformed into NEB 10-beta electrocompetent E. coli cells and plated on LB plates containing 50ug of Chloramphenicol. After overnight incubation, -2M colonies were scraped from the plate and prepped using ZymoPURE Plasmid Miniprep kit (ZYMO) to generate the final DNA library. The library was transformed into electro-competent E. coli (BW25113 DE3) harboring a selection plasmid (ColE1 , KanR). Approximately 200ng of plasmid DNA was transformed per transformation, and multiple transformations were performed to recover at least 2M clones. Transformed cells were pooled and recovered for 1 hour in 2XYT media before diluting to -1L 2XYT media containing 50ug of both Chloramphenicol and Kanamycin. This culture was incubated at 37C (first round) or 30C (second round) @150 RPM for 14 hours to enable editing of the locus. 20m L of the culture was plated across four 245mm Square BioAssay Dishes (THERMO) containing solid LB supplemented with 50ug of Chloramphenicol and Kanamycin, as well as varying amounts of Ampicillin (ranging from 0.5mg to 2mg). Colonies surviving on the plate were picked and Sanger sequenced to identify new candidates with improved precision. New TadA candidates were PCR’d and cloned into a fresh vector containing the dCas9 and sgRNA cassette and individually tested for improved survivorship compared to the parent by plating serial diluted culture on 1000ug of Ampicillin, 50ug of Chloramphenicol, and 50ug of Kanamycin. In the first round of evolution, 4 variants were recovered that improved survival compared to the ABE8e parent. No improvement in survival was observed in higher specificity mutants previously reported in the literature (N108 and F148A in both ABE8e and ABE7.10). In the second round of evolution, a single variant (8e-S7T / A114T / F148I) was recovered that outperformed its parent (8e- S7T / G67S / A114T). This variant recovered in the second round will be hereon referred as TTI.Expression and purification of TadA

[0284] E. coli BL21 STAR (DE3) competent cells (ThermoFisher Scientific) were transformed with plasmids encoding TadA-8e. The resulting expression strains were grown overnight in Luria-Bertani (LB) broth supplemented with 100 pM ZnCI2and 50 pg / mL kanamycin at 37°C. The overnight cultures were diluted 1 :100 into Terrific Broth (TB) and grown at 37°C to an ODeooof approximately 1.5. Protein expression was induced by adding 0.2 mM isopropyl p-D-1-thiogalactopyranoside (IPTG) for 5 hours. Cells were harvested by centrifugation at 4,000 g and resuspended in deaminase lysis buffer (20 mM HEPES [pH 7.5], 500 mM NaCI, 10% [v / v] glycerol, 20 mM imidazole, 1 mM TCEP) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by sonication using a Rosette Cooling Cell (Branson) with 10 s on / off pulses for a total of 10 minutes at 6 W output. The lysate was clarified by centrifugation at 20,000 g for 30 minutes at 4 °C to remove debris. The clarified lysate was loaded onto a 5 mL HisTrap FF column (Cytiva) precharged with nickel Sepharose to capture the His-tagged fusion protein. The column was washed with at least five column volumes of deaminase lysis buffer, and bound protein was eluted using the same buffer supplemented with 250 mM imidazole. The eluted protein was further purified by size exclusion chromatography (SEC) using a HiLoad 16 / 60 Superdex 200 column (Cytiva) equilibrated in 20 mM HEPES (pH 7.5), 300 mM NaCI, 10% (v / v) glycerol, and 1 mM TCEP. Deaminase-containing fractions were pooled, concentrated by ultrafiltration (Amicon-Millipore, 10 kDa molecular weight cut-off), and analyzed by SDS-PAGE to assess protein purity and yield. Typical yields were approximately 2 mg / mL.Optimized expression and purification of TadA

[0285] A modified version of TadA-8e expression and purification was initially developed for this study and later further optimized; however, the majority of the work described in this article was performed using the following conditions. E. coli BL21 STAR (DE3)- competent cells (ThermoFisher Scientific) were transformed with plasmids encoding TadA-8e. The resulting expression strains were grown overnight in Luria-Bertani (LB) broth supplemented with 100 pM ZnCI2and 50 pg / mL of kanamycin at 37 °C. Thecells were diluted 1 :100 into Terrific Broth (TB) and grown at 37 °C to OD6oo = ~1.5. The culture was cooled to 16 °C, and isopropyl -|3-d- 1 - thiogalactopyranoside (IPTG) was added at 0.5 mM to induce protein expression. After ~24 h, the cells were collected by centrifugation at 4,000 g and resuspended in deaminase lysis buffer (20 mM HEPES [pH 7.5], 300 mM NaCI, 10% [v / v] glycerol, 20 mM imidazole, 1 mM TCEP) with 1 mM phenylmethylsulfonyl fluoride. The cell supernatant was diluted two times using lysis buffer, pooled inside a Rosette Cooling Cell (Branson) and lysed by sonication (10 s pulse-on, 10 s pulse-off for 10 min total at 6 W output). The lysate supernatant was isolated following centrifugation at 20,000 g for 30 min at 4 °C to remove cellular debris and recover the supernatant. The clarified lysate was loaded onto an equilibrated 5 mL HisTrap FF column (Cytiva) prepacked with precharged nickel Sepharose to capture the His-tagged fusion protein. Subsequently, the column was washed with at least five column volumes of deaminase lysis buffer to remove non-specifically bound proteins. The His-tagged fusion protein was eluted using the deaminase lysis buffer supplemented with 250 mM imidazole. The eluted sample was next loaded onto a 5 mL HiTrap Heparin HP column (Cytiva) equilibrated in 20 mM HEPES (pH 7.5), 300 mM NaCI, and 10% (v / v) glycerol. The column was washed with at least five column volumes of equilibration buffer to remove non-specifically bound proteins before being loaded onto a HiLoad 16 / 60 S200 Superdex column (Cytiva) using an AKTA system. The protein was eluted with a linear gradient of 0.3-1 M NaCI in 20 mM HEPES and 10% (v / v) glycerol buffer (pH 7.5) containing 1 mM TCEP. Deaminase-containing fractions were pooled and concentrated by ultrafiltration (Amicon-Millipore, 10-kDa molecular weight cut-off) to ~2 mg / mL and quantified by SDS-PAGE to monitor protein purity and yield.Expression and purification of nCas9 variants

[0286] E. coli Rosetta(DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids encoding nCas9. The resulting expression strains were grown overnight in Luria-Bertani (LB) broth containing 100 pg / mL of ampicillin and 34 pg / mL chloramphenicol at 37 °C. The cells were diluted 1:100 into Terrific Broth (TB) and grown at 37 °C to OD6oo = ~1.0. The culture was cooled to 18 °C, and isopropyl -[3-d- 1- thiogalactopyranoside (IPTG) was added at 0.2 mM to induce protein expression. After ~24 h, the cells were collected by centrifugation at 4,000 g and resuspended in nickase lysis buffer (20 mM HEPES [pH 7.5], 1 M NaCI, 10% [v / v] glycerol, 10 mM imidazole, 1 mM TCEP) with 1 mM phenylmethylsulfonyl fluoride. The cells were lysedby sonication (10 s pulse-on, 10 s pulse-off for 10 min total at 6 W output) and the lysate supernatant was isolated following centrifugation at 20,000 g for 30 min at 4 °C to remove cellular debris and recover the supernatant. The clarified lysate was loaded onto an equilibrated 5 mL HisTrap FF column (Cytiva) prepacked with precharged nickel Sepharose to capture the His-tagged fusion protein. Subsequently, the column was washed with at least five column volumes of nickase lysis buffer to remove non- specifically bound proteins. The His-tagged protein eluted in nickase lysis buffer supplemented with 300 mM imidazole. The eluted sample was next loaded onto a 5 mL HiTrap Heparin HP column (Cytiva) equilibrated in 20 mM HEPES (pH 7.5), 300 mM NaCI, and 10% (v / v) glycerol. The column was washed with at least five column volumes of equilibration buffer to remove non-specifically bound proteins before being loaded onto a HiLoad 16 / 60 S200 Superdex column (Cytiva) using an AKTA system. The protein was eluted with a linear gradient of 0.3-1 M NaCI in 20 mM HEPES and 10% (v / v) glycerol buffer (pH 7.5) containing 1 mM TCEP. Nickase-containing fractions were pooled and concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cut-off) and quantified by SDS-PAGE to monitor protein purity and yield.Conjugation of TadA-8e and nCas9

[0287] Protein conjugation was performed by incubating TadA-8e:nCas9 at a molar ratio between 1:1 and 1.5:1. The reaction mixture was incubated at room temperature for 30 minutes with gentle rocking. Following incubation, the conjugated protein sample was concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cutoff) and loaded onto a HiLoad 16 / 60 S200 Superdex column (Cytiva) using an AKTA system equilibrated with gel filtration buffer (20 mM HEPES [pH 7.5], 300 mM NaCI, 10% (v / v) glycerol). Protein was loaded in volumes no greater than 2 mL. Fractions containing the conjugated protein complex were pooled and concentrated for further analysis (Amicon-Millipore, 100-kDa molecular weight cut-off). Purified proteins were concentrated to ~50 pM, filtered using Spin-X centrifuge tube (0.22 pM, Corning) and stored at -80 °C.Cas9 sgRNA

[0288] S. pyogenes Cas9 gRNAs were purchased with manufacturer-recommended standard chemical modifications from Synthego and resuspended in water, or from IDT(Alt-R) and resuspended in IDT duplex buffer or diethyl pyrocarbonate (DEPC)-treated water. Before use, sgRNAs were diluted to 30 pM in 20 mM HEPES pH 7.5 and 150 mM NaCI, then refolded by warming to 95 °C for 5 min and slow cooling to room temperature for 25 min.RNP formation

[0289] For electroporation and peptide-mediated delivery experiments, Cas9 protein was diluted to 20 pM in ‘RNP buffer’ (20 mM HEPES pH 7.5, 150 mM NaCI, 10% glycerol and 2 mM MgCI2). The sgRNA was diluted to 30 pM in 20 mM HEPES pH 7.5, 150 mM NaCI, 10% glycerol and 2 mM MgCI2. The molar ratio of Cas9:sgRNA was 1 :1.5 unless otherwise specified. Cas9 was mixed with sgRNA in equal volumes yielding 10 pM RNP complex, with the RNP concentration defined by the amount of Cas9 protein.Peptide and RNP delivery formulations in T cells

[0290] Peptides (10 mM in 100% DMSO) were diluted in DEPC-treated water to 1 mM (resulting in a solution of 90% water and 10% DMSO) and added to RNP, resulting in a volume no greater than 20% of the eventual final volume (for example, <20 pl formulation for a well with 100 pl cells in Opti-MEM). The RNP / peptide mixture was added to a 96-well round-bottom plate, and 200 x 103cells in 100 pl Opti-MEM (Gibco) per well was added to the RNP / peptide mixture. The final dose of RNP during cell treatment was 50 pmol per well with a final peptide concentration of 10 pM, unless stated otherwise. In all cases of peptide-enabled delivery, the final concentration of DMSO was proportional to the peptide concentration: 0.1% DMSO per 10 pM peptide; this concentration of DMSO was used for the ‘mock’ negative control conditions. After a 1 h incubation at 37 °C, 100 pl of treated volume was split in half into two plates, then 150 pl culture medium was added per well, thus diluting but not removing the treatment. The concentration of additives and stimulation cocktail in the recovery medium was such that the final concentrations matched the description above for each cell type.RNP electroporation in T cells

[0291] In a 4D nucleofector (Lonza), 20 pmol of Cas9 RNP was electroporated into 200 x 1O3T cells resuspended in 20 pl of P3 buffer and supplement (Lonza V4XP- 3032) using the EH-115 pulse code. Cells were incubated for 10 min at 37 °C, thenrescued with 80 pl of pre-warmed culture medium before diluting for further cell culture as above.Flow cytometry

[0292] Flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific) or on an LSRFortessa X-50 flow cytometer (BD Biosciences). Cells were resuspended in FACS buffer (phosphate-buffered saline (PBS), 2% FBS and 1 mM EDTA) and stained with live-dead stain and surface marker-targeting antibodies according to manufacturer-provided instructions. Sampling was at defined volumes (60 pl per well) to quantify cell counts. Cytometry data were processed and analyzed using FlowJo software (BD Biosciences). mRNA preparation and delivery to CD34+ HSPCs

[0293] Human CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from healthy donors (n = 3) using the EasySep™ Human CD34 Positive Selection Kit II (STEMCELL Technologies). Cells were expanded in StemSpan medium supplemented with the recommended cytokines prior to electroporation. For base editing experiments, ~0.3 10A6 cells per reaction were electroporated using a MaxCyte GTx instrument in an OC-25 cuvette with the HSC3 program. Two modalities were compared: (i) mRNA delivery, where 5 pg of in vitro transcribed BE mRNA was co-electroporated with 100 pmol of sgRNA, and (ii) protein delivery, where 100 pmol of purified BE protein was pre-complexed with 300 pmol of sgRNA for 15 min at room temperature before nucleofection. sgRNAs were purchased from Synthego.

[0294] BE mRNA was synthesized using the Takara IVTpro™ T7 mRNA Synthesis Kit (Cat. #6144). To enhance stability and translational efficiency, pseudouridine-5'- triphosphate (pseudo-UTP; Jena Bioscience, Cat. #NU-1139L) was incorporated in place of DTP, and transcripts were capped with mA7G(5')ppp(5')G ARCA cap analog (Jena Bioscience, Cat. #NU-855L).

[0295] Cell viability and recovery were assessed 48 h post-electroporation. Genomic DNA was extracted from unedited control (UE) and targeted edited (G11) samples at multiple time points: day 3 (D3), day 7 (D7), and day 11 (D11) post-electroporation. DNA was isolated using a rapid extraction protocol for D3 and D7 samples, and a column-based method for D11 samples. The latter samples were subjected to nextgeneration sequencing (NGS) to confirm final editing efficiencies. At each time point, parallel cell aliquots were cryopreserved (D3: expansion phase; D7: day 4 of Phase Idifferentiation; D11 : day 8 of Phase I differentiation). Editing efficiency was initially determined by Sanger sequencing, with chromatograms analyzed using EditR.Brain innage analysis

[0296] Neuronal genome editing from 3-channel TIFF stacks (DAPI, NeuN-Red, GFP) was quantified using a MATLAB pipeline with user-drawn, striatal- ROIs. Nuclei were segmented on DAPI by global thresholding and connected-component labeling, and neuron identity was assigned when NeuN mean intensity at the nuclear centroid exceeded background. To capture the spatial extent of transduction, a GFP distribution map was generated by applying an adaptive global threshold (Otsu on ROI-restricted data) followed by morphological opening, small-object removal, gapbridging, closing, and convex-hull union over connected components to span contiguous domains. For binarization of GFP+ / NeuN+ cells, GFP positivity was assessed with the threshold-only core mask, labeling a neuron GFP-positive when >50% of its nuclear segment overlapped this core. Report here: (i) percent striatum edited, computed as the volume fraction of GFP+ tissue within each striatum as defined by the distribution map, and (ii) percent neuronal editing within the edited region, computed as the fraction of NeuN-positive nuclei whose centroids fall inside the distribution map that meet the >50% GFP overlap criteria.Confocal Scanning Laser Microscopy

[0297] Coronal brain sections were mounted and stained on poly-L-lysine-coated slides. Sections were incubated with NeuroTrace™ far-red per the manufacturer’s instructions, counterstained with DAPI, rinsed, and mounted in antifade under #1.5 coverslips. Images were acquired on a Zeiss LSM 990 using a Plan-Apochromat 20* objective with sequential channel acquisition (DAPI 405 nm, GFP 488 nm, NeuroTrace far-red 633 / 640 nm). The pinhole was referenced to the shortest- wavelength channel; laser power and detector gain were adjusted to avoid saturation and held constant across samples.Whole-section imaging

[0298] For whole-slide maps, adjacent sections mounted on glass were immunolabeled with anti-NeuN (standard block, primary incubation, fluorophore-conjugated secondary), counterstained with DAPI, and cover-slipped in antifade. Slides were imaged on a Zeiss Axio Scan 7 using a 20x objective with autofocus and sequential fluorescenceacquisition. Exposure / gain were standardized across slides, and images were exported as 16-bit multichannel pyramidal TIFFs.Example 6: Expression of both components in the same cell

[0299] FIG. 11. The BEaST system is functional when two distinct plasmids are used for expression of components in the same cell - in this case a single population of E. coli cells. This SDS-PAGE gel (coomassie stained) shows partial purification of BEaST ABE expressed in a single population of E. coli cells following transformation using two plasmids: one encoding a tandem deaminase gene with a C-terminal SpyTag, the other encoding a Cas9 nickase gene with an N-terminal SpyCatcher tag. Using a nickel affinity column, the histidine-tagged BEaST ABE protein was eluted (lane 4), demonstrating that the BEaST polypeptides can assemble within the E. coli cell (or in cell lysate) following expression using two plasmids in the same cell population.

[0300] FIG 12. Plasmid map diagram representing the encoded BEaST system components on a single DNA: in this example embodiment, two tandem ORFs encoding (i) tandem deaminase gene (labeled as “TadA dimer”) with a C-terminal SpyTag, followed by (ii) a Cas9 nickase gene with an N-terminal SpyCatcher tag. Each gene has its own T7 promoter and lac operator with one transcription terminator present at the 3’ end of the Cas9 gene. This plasmid construct allows for two separate transcripts to be produced. The first transcript includes both TadA dimer and Cas9 (resulting in two polypeptides following translation); the second transcript only includes Cas9 (one polypeptide following translation).

[0301] FIG 13. The BEaST system is functional when a single DNA (e.g., plasmid) is used for expression of BEaST components in a single population of E. coli cells. This SDS- PAGE gel (coomassie stained) shows evidence of assembled BEaST ABE resulting from component expression in a single population of E. coli cells following transformation using one plasmid encoding both (i) a tandem deaminase gene with a C-terminal SpyTag and (ii) a Cas9 nickase gene with an N-terminal SpyCatcher tag. This demonstrates that the BEaST polypeptides can assemble within the E. coli cell (or in cell lysate) following component expression using a single plasmid.

[0302] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certainchanges and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0303] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0304] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

CLAIMSWhat is claimed is:

1. A programmable base editor, comprising: a CRISPR-Cas fusion protein comprising a CRISPR-Cas effector protein fused to a first member of a split-protein binding pair, wherein the CRISPR-Cas effector protein is a nickase or is catalytically inactive, and a deaminase fusion protein comprising a deaminase protein fused to a second member of the split-protein binding pair, wherein the split-protein binding pair comprises a catcher / tag system or a GFP1- 10 / GFP11 system.

2. The programmable base editor of claim 1 , wherein one member of the split-protein binding pair is a catcher protein, and the other member is a partner tag protein, and wherein the CRISPR-Cas fusion protein is covalently linked to the deaminase fusion protein via a post- translational isopeptide bond between the catcher protein and the partner tag protein.

3. The programmable base editor of claim 2, wherein the catcher protein is SpyCatcherOOl , SpyCatcher AN 1 AC 1 , SpyLigase, SnoopCatcher, SpyCatcher002, SpyCatcher003, SnoopLigase, or SpyDock.

4. The programmable base editor of claim 2, wherein the catcher protein comprises SEQ ID NO: 11.

5. The programmable base editor of claim 2 or claim 3, wherein the partner tag protein is comprises SpyTag (AHIVMVDAYKPTK; SEQ ID NO: 1), KTag (ATHIKFSKRD; SEQ ID NO: 2), SnoopTag (KLGDIEFIKVNK; SEQ ID NO: 3), SpyTag002 (VPTIVMVDAYKRYK; SEQ ID NO: 4), SnoopTagJr (KLGSIEFIKVNK; SEQ ID NO: 5), DogTag (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 6), or SpyTag003 (RGVPHIVMVDAYKRYK) (SEQ ID NO: 7).

6. The programmable base editor of any one of claims 2-5, wherein the partner tag protein comprises RGVPHIVMVDAYKRYK (SEQ ID NO: 7).

7. The programmable base editor of any one of claims 1-6, wherein the deaminase protein is an adenosine deaminase.

8. The programmable base editor of any one of claims 1-6, wherein the deaminase protein is a cytidine deaminase.

9. The programmable base editor of any one of claims 1-6, wherein the deaminase protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of any one of SEQ ID NOs: 32-35 and 74-75.

10. The programmable base editor of any one of claims 1-6, wherein the deaminase protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 32.11 . The programmable base editor of any one of claims 1-6, wherein the deaminase protein is TadA-8e.

12. The programmable base editor of any one of claims 1-11 , wherein the CRISPR-Cas effector protein is an nCas9, dCas9, or dCas12a protein.

13. The programmable base editor of any one of claims 1-11 , wherein the CRISPR-Cas effector protein is an nCas9 protein.

14. The programmable base editor of any one of claims 1-11 , wherein the CRISPR-Cas effector protein is a dCas9 or dCas12a protein.

15. The programmable base editor of any one of claims 1-14, wherein the first member of the split-protein binding pair is fused N-terminal to the CRISPR-Cas effector protein.

16. The programmable base editor of any one of claims 1-15, wherein the second member of the split-protein binding pair is fused C-terminal to the deaminase protein.

17. The programmable base editor of any one of claims 1-16, comprising one or more nuclear localization signals (NLSs).

18. The programmable base editor of any one of claims 2-17, wherein the CRISPR-Cas effector protein is fused to the catcher protein, and the deaminase protein is fused to the partner tag protein.

19. The programmable base editor of claim 18, wherein: the catcher protein is fused N-terminal to the CRISPR-Cas effector protein, the catcher protein comprises SEQ ID NO: 11 , and the CRISPR-Cas effector protein is a nickase Cas9 protein (nCas9); and the partner tag protein is fused C-terminal to the deaminase protein, the partner tag protein comprises RGVPHIVMVDAYKRYK (SEQ ID NO: 7), and the deaminase protein is Tad- 8e.

20. A composition comprising the programmable base editor of any one of claims 1-19, and a guide RNA.

21. A method of producing the programmable base editor of any one of claims 1-19, comprising contacting the CRISPR-Cas fusion protein with the deaminase fusion protein, thus producing the programmable base editor.

22. The method of claim 21 , wherein the split-protein binding pair comprises the catcher / tag system, wherein one member of the split-protein binding pair is a catcher protein, and the other member is a partner tag protein, and wherein said contacting results in covalent linkage of the CRISPR-Cas fusion protein with the deaminase fusion protein via post-translational isopeptide bond formation between the catcher protein and the partner tag protein.

23. The method of claim 20 or claim 21 , wherein said contacting takes place at a pH of about 7.5.

24. The method of any one of claims 21-23, wherein said contacting takes place at a salt concentration of about 300mM NaCI.

25. The method of any one of claims 21-24, further comprising, prior to said contacting, producing the CRISPR-Cas fusion protein and the deaminase fusion protein.

26. The method of claim 25, wherein said producing comprises expressing the CRISPR- Cas fusion protein and the deaminase fusion protein separately in cells, and extracting the CRISPR-Cas fusion protein and the deaminase fusion protein from the cells.

27. The method of claim 25 or claim 26, wherein said producing comprises purification of the CRISPR-Cas fusion protein using affinity chromatography and gel filtration, and purification of the deaminase fusion protein using affinity chromatography.

28. The method of any one of claims 25-27, wherein the CRISPR-Cas fusion protein and the deaminase fusion protein are purified at a pH of about 7.5.

29. The method of any one of claims 21-28, further comprising, after said contacting, performing one or more purification steps to enrich for the programmable base editor.

30. The method of claim 29, wherein said one or more purification steps comprise gel filtration.31 . The method of any one of claims 21-30, wherein the programmable base editor that is produced retains 80% or more deaminase activity when compared to the deaminase activity of the deaminase fusion protein prior to contact with the CRISPR-Cas fusion protein.

32. A method for modifying a target nucleic acid, the method comprising: contacting a target nucleic acid with the programmable base editor of any of claims 1-18, and a guide RNA, wherein the guide RNA hybridizes to a target sequence in the target nucleic acid, and wherein said contacting results in the deamination of an adenine or a cytosine of the target nucleic acid.

33. The method of claim 32, wherein the target nucleic acid is genomic DNA.

34. The method of any one claim 32 or 33, wherein the target sequence is a protein-coding sequence.

35. The method of any one of claims 32-34, wherein said contacting takes place inside of a cell that comprises the target nucleic acid.

36. The method of claim 35, wherein said contacting comprising introducing into the cell, (i) the programmable base editor, and (ii) the guide RNA or a nucleic acid encoding the guide RNA.

37. The method of claim 35 or claim 36, wherein the cell is a eukaryotic cell.

38. The method of claim 35 or claim 36, wherein the cell is a mammalian cell.

39. The method of any of claims 35-38, wherein the cell is in vitro.

40. The method of any of claims 35-38, wherein the cell is in vivo.41 . The method of any of claims 36-40, wherein the programmable base editor and the guide RNA are introduced into the cell using a viral vector or a nanoparticle.

42. The method of claim 41 , wherein the nanoparticle is a lipid nanoparticle.

43. A nucleic acid system comprising:(a) a first nucleotide sequence encoding the CRISPR-Cas fusion protein of any one of claims 1-19, or(b) a second nucleotide sequence encoding the deaminase fusion protein of any one of claims 1-19, or(c) both (a) and (b).

44. The nucleic acid system of claim 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in a prokaryotic cell.

45. The nucleic acid system of claim 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in an E. coli cell.

46. The nucleic acid system of claim 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in a eukaryotic cell.

47. The nucleic acid system of claim 43, wherein the first nucleotide sequence and / or the second nucleotide sequence is codon optimized for expression in a mammalian cell.

48. The nucleic acid system of any one of claims 43-47, comprising a first nucleic acid comprising the first nucleotide sequence, and a second nucleic acid comprising the second nucleotide sequence.

49. The nucleic acid system of claim 48, wherein the first and / or second nucleic acid is a plasmid.

50. The nucleic acid system of claim 48, wherein the first and / or second nucleic acid is a viral vector.51 . The nucleic acid system of any one of claims 43-47, comprising a nucleic acid molecule that comprises both the first nucleotide sequence and the second nucleotide sequence.

52. The nucleic acid system of claim 51 , wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters.

53. The nucleic acid system of claim 51 or claim 52, wherein the nucleic acid molecule is a plasmid or a viral vector.

54. The method of any one of claims 25 and 29-31 , wherein said producing comprises expressing the CRISPR-Cas fusion protein and the deaminase fusion protein in the same cell, thus leading to contact of the CRISPR-Cas fusion protein with the deaminase fusion protein and therefore formation of the programmable base editor in the same cell.

55. The method of claim 54, wherein the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by and expressed from the same nucleic acid molecule.

56. The method of claim 54, wherein the CRISPR-Cas fusion protein and the deaminase fusion protein are encoded by and expressed from the same plasmid.

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