WO2020181178A1 - T:a to a:t base editing through thymine alkylation - Google Patents

Abstract

The present disclosure provides for base editors which satisfy a need in the art for installation of targeted transversions of thymine (T) to adenine (A), or correspondingly, transversions of adenine (A) to thymine (T). The nucleobase editor domains include a nucleic acid programmable DNA binding protein and a thymine alkyltransferase domain, engineered through the use of continuous or non-continuous evolution systems, such as phage-assisted continuous evolution (PACE). In particular, the present disclosure provides for thymine-to-adenine (or adenine-to-thymine) base editor variants which satisfy deficiencies in the art for base editors that can install A:T to T:A single-base transversion mutations. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, pharmaceutical compositions comprising, and vectors and kits for the generation of, targeted base editors are provided. In some embodiments, cells containing such vectors are provided. In some embodiments, methods of treatment comprising administering the base editors are provided.

Description

T:A to A:T BASE EDITING THROUGH THYMINE ALKYLATION

RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 62/814,798, filed on March 6, 2019, the entire disclosure of which is incorporated by reference herein.

BACKGROUND OF THE DISCLOSURE

[0002] Targeted editing of nucleic acid sequences, including the targeted cleavage or targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for genetic diseases, including those caused by point mutations. Point mutations represent the majority of known human genetic variants associated with disease. Developing robust methods to introduce and correct point mutations is therefore important in understanding and treating diseases with a genetic component.

[0003] Base editing involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. For certain approaches, this can be achieved without requiring double-stranded DNA breaks (DSB). Since many genetic diseases arise from point mutations, this technology has important implications in the study of human health and disease. Engineered base editors are capable of editing many targets with high efficiency, often achieving editing of 30-70% of cells following a single treatment, without selective enrichment of the cell population for editing events.

SUMMARY OF THE DISCLOSURE

[0004] Engineered base editors have been recently developed. Reference is made to Komor, A.C. et al, Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity, Sci Adv 3 (2017); Rees, H.A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790 (2017); U.S. Patent Publication No. 2018/0073012, published March 15, 2018, U.S. Patent Publication No.

2017/0121693, published May 4, 2017, International Publication No. WO 2017/070633, published April 27, 2017, and U.S. Patent Publication No. 2015/0166980, published June 18, 2015, U.S. Patent No. 9,840,699, issued December 12, 2017, U.S. Patent No. 10,077,453, issued September 18, 2018, and International Application No. PCT/US2019/61685, filed November 15, 2019, the contents of each of which are incorporated herein by reference in their entireties, each of which are incorporated herein in their entireties. Base editors (BEs) are typically fusions of a Cas (“CRISPR-associated”) domain and a nucleobase modification domain (e.g., a natural or evolved deaminase, such as a cytidine deaminase that include APOBEC1 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1”), CDA (“cytidine deaminase”), and ATP (“activation-induced cytidine deaminase”)) domains. In some cases, base editors may also include proteins or domains that alter cellular DNA repair processes to increase the efficiency and/or stability of the resulting single-nucleotide change.

[0005] Two classes of base editors have been generally described to date: cytidine base editors convert target C:G base pairs to T:A base pairs, and adenine base editors convert A:T base pairs to G:C base pairs. Collectively, these two classes of base editors enable the targeted installation of all possible transition mutations (C-to-T, G-to-A, A-to-G, T-to-C, C- to-U, and A-to-U), which collectively account for about 61% of known human pathogenic small nucleotide polymorphisms (SNPs) in the ClinVar database. See Gaudelli, N.M. el al, Programmable base editing of A:T to G:C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017). In particular, C-to-T base editors use a cytidine deaminase to convert cytidine to uridine in the single-stranded DNA loop opened by Cas9 (“CRISPR-associated protein 9”). The opposite strand is nicked by Cas9 to stimulate DNA repair mechanisms that use the edited strand as a template, while a fused uracil glycosylase inhibitor slows excision of the edited base. Eventually, DNA repair leads to a C:G to T:A base pair conversion. This class of base editor is described in U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued on January 1, 2019, as U.S. Patent No. 10,167,457, which is incorporated by reference in its entirety herein.

[0006] A major limitation of base editing is the inability to generate transversion (purine <- pyrimidine) changes, which are needed to correct -38% of known human pathogenic SNPs. See Komor, A.C. et al, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature 533, 420-424 (2016) and Landrum, M.J. et al, ClinVar: public archive of relationships among sequence variation and human phenotype, Nucleic Acids Res. 42, D980-985 (2014), each of which is incorporated by reference.

Currently, transversions can only be repaired by nuclease-mediated formation of a double- stranded break (DSB) followed by homology directed repair (HDR), which is typically inefficient, especially in non-mitotic cells, and leads to undesired byproducts such as indels (insertions and deletions) and translocations. See Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes, Cell 168, 20-36, (2017), which is herein incorporated by reference. Since nucleobase deamination alone cannot interconvert purines and pyrimidines, the development of transversion base editors requires the development of a new editing strategy, such as the manipulation of endogenous DNA repair pathways or a different nucleobase chemical transformation. The present disclosure describes novel transversion base editors using an innovative strategy. These base editorsgreatly expand the capabilities of base editing.

[0007] The present disclosure provides transversion base editors which add to the repertoire of base editors that have already been developed. In particular, the present disclosure provides for thymine-to-adenine or“TABE” (or adenine-to-thymine or“ATBE”) transversion base editors which satisfy the need in the art for the installation of targeted single-base transversion nucleobase changes in a target nucleotide sequence, e.g., a genome. In addition, the present disclosure provides for nucleic acid molecules encoding and/or expressing the thymine-to-adenine and adenine-to-thymine transversion base editors described herein, as well as expression vectors or constructs for expressing these transversion base editors, host cells comprising said nucleic acid molecules and expression vectors, and compositions for delivering and/or administering nucleic acid-based embodiments described herein. In addition, the disclosure provides for compositions comprising these transversion base editors. Still further, the present disclosure provides for methods of making the transversion base editors, as well as methods of using the transversion base editors or nucleic acid molecules encoding such transversion base editors in applications including editing a nucleic acid molecule, e.g., a genome.

[0008] The present inventors have developed novel transversion base editors, and in particular a novel base editor that installs a T-to-A transversion in a targeted manner, through a thymine alkylation reaction. This new strategy allows for the efficient and specific transversion of A-to-T or T-to-A using the inventive base editors described

herein. Specifically, the enzyme-catalyzed addition of an alkyl group, e.g. a methyl or carboxymethyl group, to a targeted thymine (T) in a nucleic acid of interest is induced, resulting in N3-methyl thymine or N3 -carboxymethyl thymine formation, respectively. In another embodiment, the enzyme-catalyzed addition of a 3-amino-3-carboxypropyl group to a targeted T in a nucleic acid of interest is induced, resulting in the formation of an N3-3- amino-3-carboxypropyl thymine. Each of N3-methyl thymine, N3 -carboxymethyl thymine, and N3-3-amino-3-carboxypropyl thymine disrupts the hydrogen bonding interactions with the base-paired adenine of the unmutated strand. Without wishing to be bound by any particular theory, the cell’s replication machinery interprets the alkylated thymine as an adenine and converts the mismatched adenine of the unmutated strand to a thymine. During a subsequent round of replication or mismatch repair, the alkylated thymine is converted to an adenine. A desired T-to-A transversion is thus achieved. Thymine alkylation is achieved by the targeted use of a fusion protein comprising a Cas9 (e.g., a catalytically dead Cas9 (“dCas9”) or Cas9 nickase (“nCas9”)) domain, a thymine alkyltransferase domain, and optionally a linker connecting these two domains (see FIGs. 1A, 2A).

[0009] The nucleic acid programmable DNA binding protein (napDNAbp) may be a Cas9 domain. The napDNAbp may also be a CasX, a CasY, a C2cl, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Casl2a (formerly known as Cpfl), a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, an LbCasl2a, an AsCasl2a, a Cas9-KKH, a circularly permuted Cas9, an Argonaute (Ago), a SmacCas9, or a Spy-macCas9. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 (dCas9) domain, or a Cas9 nickase (nCas9) domain. Further, for all strategies, the domains of the base editor fusion protein maye be interconnected with a linker. This linker may be any suitable amino acid linker, synthetic linker, polymer, or a covalent bond.

Exemplary linkers include any of the following amino acid sequences:

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5); SGGSGGSGGS (SEQ ID NO: 6); GGG; GGGS (SEQ ID NO: 10); SGGGS (SEQ ID NO: 1);

SGSETPGTSESATPES (SEQ ID NO: 49); or SGGS (SEQ ID NO: 8).

[0010] The disclosure provides a base editor fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) a thymine alkyltransferase. In various embodiments of the base editor fusion proteins, the thymine alkyltransferase is a wild-type thymine alkyltransferase. In certain embodiments, the thymine alkyltransferase is a wild-type RsmE, or a variant thereof, that methylates a thymine in a nucleic acid. In certain embodiments, the RsmE is an Escherichia coli RsmE, or a variant thereof. In certain embodiments, the RsmE variant has been evolved to accept a modified 5-adcnosyl- methionine (SAM) cofactor, e.g. a SAM cofactor modified to include a carboxymethyl domain on the S⁺ center, so as to yield an N3 -carboxymethyl thymine product.

[0011] In various embodiments, the thymine alkyltransferase comprises any one of the amino acid sequences of SEQ ID NOs: 50-65. In various embodiments, the thymine

alkyltransferase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,

98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 50-65. In particular embodiments, the thymine alkyltransferase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 62-65. In particular embodiments, the thymine alkyltransferase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 51.

[0012] In certain embodiments, the base editor fusion protein further comprises an inhibitor of DNA alkylation repair (“iDAR”) that may covalently or non-covalently bind to a mutated nucleobase to prevent its excision during subsequent mismatch repair or oxidative repair.

Use of an iDAR in the base editor fusion protein may increase base editing efficiency for the thymine alkylation and other alkylation strategies. In certain embodiments, the iDAR comprises a catalytically inactive glycosylase or catalytically inactive dioxygenase that binds N3-methyl thymine, N3-carboxymethyl thymine, or N3-3-amino-3-carboxypropyl thymine to prevent its excision during subsequent mismatch repair or oxidative repair.

[0013] In various embodiments, the base editor fusion proteins described herein may comprise any of the following structures: NH2-[napDNAbp]-[thymine alkyltransferase] - COOH; or NH2-[thymine alkyltransferase]-[napDNAbp]-COOH; wherein each instance of “]-[” comprises an optional linker.

[0014] In various embodiments, when the fusion proteins include an iDAR domain, the base editor fusion proteins described herein can may comprise any of the following structures: Nth- [iDAR] - [napDNAbp] - [thymine alkyltransferase] -COOH; N¾- [napDNAbp] - [iDAR] - [ thymine alkyltransferase] -COOH; NH2-[napDNAbp]-[thymine alkyltransferase] -[iDAR] - COOH; NH2-[iDAR]-[ thymine alkyltransferase ]- [napDNAbp] -COOH; NH2-[thymine alkyltransferase]-[iDAR]-[napDNAbp]-COOH; or NH2-[thymine alkyltransferase] - [napDNAbp]-[iDAR]-COOH; wherein each instance of“]-[” comprises an optional linker.

[0015] In various other embodiments, the disclosure provides nucleic acid molecules or constructs encoding any of the base editor fusion proteins, or domains thereof. The nucleic acid sequences may be codon-optimized for expression in the cells of any organism of interest. In certain embodiments, the nucleic acid sequence is codon-optimized for expression in human cells.

[0016] In other embodiments, the disclosure provides polynucleotides and/or vectors encoding any of the base editor fusion proteins described herein, or domains thereof. These nucleic acid sequences are typically engineered or modified experimentally. For instance, these nucleic acid sequences may be codon-optimized for expression in an organism of interest, e.g. mammalian cells. In certain embodiments, the nucleic acid sequences are codon-optimized for expression in human cells. In other embodiments, cells containing such polynucleotides or constructs are provided. In other embodiments, complexes comprising any of the fusion proteins described herein and a guide RNA bound to the napDNAbp domain of the fusion protein are provided.

[0017] In other embodiments, the disclosure provides a pharmaceutical composition comprising any of the fusion proteins described herein and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition further comprises a gRNA. In other embodiments, the disclosure provides a kit comprising a nucleic acid construct that includes (i) a nucleic acid sequence encoding any of the fusion proteins and gRNAs described herein; (ii) a heterologous promoter that drives expression of the sequence of (i); and optionally an expression construct encoding a guide RNA backbone and the target sequence.

[0018] In some embodiments, methods for targeted nucleic acid editing are provided. The methods described herein typically comprise i) contacting a nucleic acidsequence with a complex comprising any of the fusion proteins described herein and a guide nucleic acid, wherein the double-stranded DNA comprises a target A:T (or T:A) nucleobase pair, and ii) editing the thymine (or adenine) of the A:T (or T:A) nucleobase pair. The methods may further comprise iii) cutting or nicking the non-edited strand of the double- stranded DNA.

[0019] In some embodiments, methods of treatment using the inventive base editors are provided. The methods described herein may comprise treating a subject having or at risk of developing a disease, disorder, or condition, comprising administering to the subject a fusion protein as described herein, a polynucleotide as described herein, a vector as described herein, or a pharmaceutical composition as described herein.

[0020] It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non limiting embodiments when considered in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] FIG. 1A is a schematic illustration showing an exemplary fusion protein of the disclosure. A fusion protein comprising a dCas9 linked to a thymine alkyltransferase enzyme is targeted to the correct thymine nucleobase through the hybridization of a single-guide RNA (“sgRNA”) to a complementary sequence of a nucleic acid. The thymine

alkyltransferase alkylates the thymine to an N3-methylthymine, and subsequently, the cell’s native replication/repair machinery recognizes the mutated base and effects the desired change to an adenine nucleobase. Abbreviations: m3T, N3-methylthymine; iDAR, inhibitor of DNA alkylation repair; sgRNA, single-guide RNA; PAM, protospacer adjacent motif.

[0023] FIG. IB depicts the chemical conversion of thymine to N3-methylthymine (or N3- carboxymethylthymine), which disrupts existing hydrogen bonding with the adenine of the unmutated strand. Without wishing to be bound by any particular theory, the cell’s replication machinery interprets the alkylated thymine as an adenine (A), and converts the mismatched adenine to an thymine. During a subsequent replication-and-repair cycle, the alkylated thymine is converted to an adenosine, completing the desired T:A to A:T mutation.

[0024] FIG. 2A is a schematic illustration showing a second exemplary fusion protein of the disclosure. A fusion protein comprising a dCas9 linked to a aminocarboxypropyl transferase enzyme is targeted to the correct thymine nucleobase through the hybridization of a single guide RNA (“sgRNA”) to a complementary sequence of a nucleic acid. The

aminocarboxypropyl transferase alkylates the thymine with an aminocarboxpropyl moiety to form an N3-3-amino-3-carboxypropyl thymine product, and subsequently, the cell’s native replication/repair machinery recognizes the mutated base and effects the desired change to an adenine nucleobase. Abbreviations: Acp3T, N3-3-amino-3-carboxypropyl thymine.

[0025] FIG. 2B depicts the chemical conversion of thymine to N3-3-amino-3-carboxypropyl thymine. Without wishing to be bound by any particular theory, the cell’s replication machinery interprets the alkylated thymine as an adenine (A), and converts the mismatched adenine to an thymine. During a subsequent replication-and-repair cycle, the alkylated thymine is converted to an adenosine, completing the desired T:A to A:T mutation.

[0026] FIG. 3 depicts an exemplary assay for selection of evolved variants of E. coli RsmE rRNA methyltransferase that are highly effective at methylating thymine. Libraries of mutagenized RsmE-dCas9 fusion proteins, targeting guide RNAs, and a selection plasmid containing an inactivated spectinomycin resistance gene with mutations at the active site (D182V or K205T) that require T-to-A editing to correct, are transformed into E. coli cells, which are plated onto agar media containing spectinomycin and sucrose. Cells harboring plasmids with RsmE mutants that restore antibiotic resistance are isolated and subjected to further rounds of mutation and selection under varying selection stringencies. RsmE variants emerging from each round of selection are then expressed within a fusion construct comprising a Cas9 nickase (nCas9). The resulting fusion proteins are tested for base editing activity in mammalian cells.

DEFINITIONS

[0027] As used herein and in the claims, the singular forms“a,”“an,” and“the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to“an agent” includes a single agent and a plurality of such agents.

[0028] The term“accessory plasmid,” as used herein, refers to a plasmid comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter. In the context of continuous evolution of genes, transcription from the conditional promoter of the accessory plasmid is typically activated, directly or indirectly, by a function of the gene to be evolved. Accordingly, the accessory plasmid serves the function of conveying a competitive advantage to those viral vectors in a given population of viral vectors that carry a version of the gene to be evolved able to activate the conditional promoter or able to activate the conditional promoter more strongly than other versions of the gene to be evolved. In some embodiments, only viral vectors carrying an“activating” version of the gene to be evolved will be able to induce expression of the gene required to generate infectious viral particles in the host cell, and, thus, allow for packaging and propagation of the viral genome in the flow of host cells. Vectors carrying non-activating versions of the gene to be evolved, on the other hand, will not induce expression of the gene required to generate infectious viral vectors, and, thus, will not be packaged into viral particles that can infect fresh host cells. Exemplary accessory plasmids have been described, for example in U.S. Application No. 15/567,312, published as U.S. Pub. No. 2018/0087046, filed on April 15, 2016, the entire contents of which is incorporated by reference.

[0029]“Base editing” is a genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain embodiments, this can be achieved without requiring double- stranded DNA breaks (DSB). To date, other genome editing techniques, including CRISPR-based systems, begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low (e.g., typically 0.1% to 5%), with the major genome editing products being indels. In order to increase the efficiency of gene correction without simultaneously introducing random indels, the CRISPR/Cas9 system was previously modified to convert directly one DNA base into another without DSB formation. See, Komor, A.C., et al, Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage. Nature 533, 420-424 (2016), the entire contents of which is incorporated by reference herein.

[0030] In principle, there are 12 possible base-to-base changes that may occur via individual or sequential use of transition (i.e., a purine-to-purine change or pyrimidine-to-pyrimidine change) or transversion (i.e., a purine-to-pyrimidine or pyrimidine-to-purine) editors. These include:

• Transition base editors:

o C-to-T base editor (or“CTBE”). This type of editor converts a C:G Watson-Crick nucleobase pair to a T:A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a G-to-A base editor (or“GABE”).

o A-to-G base editor (or“AGBE”). This type of editor converts a A:T Watson-Crick nucleobase pair to a G:C Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a T-to-C base editor (or“TCBE”).

• Transversion base editors:

o C-to-G base editor (or“CGBE”). This type of editor converts a C:G Watson-Crick nucleobase pair to a G:C Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a G-to-C base editor (or“GCBE”).

o A-to-C base editor (or“ACBE”). This type of editor converts a A:T Watson-Crick nucleobase pair to a C:G Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a T-to-G base editor (or“TGBE”).

o G-to-T base editor (or“GTBE”). This type of editor converts a G:C Watson-Crick nucleobase pair to a T:A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also

interchanged as a result of the conversion, this category of base editor may also be referred to as a C-to-A base editor (or“CABE”).

o A-to-T base editor (or“ATBE”). This type of editor converts a A:T Watson-Crick nucleobase pair to a T:A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a T-to-A base editor (or“TABE”).

[0031] The term“base editors (BEs)”, as used herein, refers to the Cas-fusion proteins described herein. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) fused to a thymine alkyltransferase which binds a nucleic acid in a guide RNA- programmed manner via the formation of an R-loop, but does not cleave the nucleic acid.

For example, the dCas9 domain of the fusion protein may include a D10A and a H840A mutation (which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex), as described in PCT/US2016/058344 (filed on October 22, 2016 and published as WO 2017/070632 on April 27, 2017), which is incorporated herein by reference in its entirety.

The DNA cleavage domain of S. pyogenes Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand

complementary to the gRNA (the“targeted strand,” or the strand at which editing or oxidation occurs), whereas the RuvCl subdomain cleaves the non-complementary strand containing the PAM sequence (the“non-targeted strand”, or the strand at which editing or alkylation does not occur). The RuvCl mutant D10A generates a nick on the targeted strand, while the HNH mutant H840A generates a nick on the non-targeted strand (see Jinek et al, Science. 337:816-821(2012); Qi et al, Cell. 28; 152(5): 1173-83 (2013)).

[0032] In some embodiments, the fusion protein comprises a Cas9 nickase fused to a thymine alkyltransferase, e.g., a thymine methyltransferase which converts a thymine nucleobase to N3-methylthymine. The term“base editors” encompasses the base editors described herein as well as any base editor known or described in the art at the time of this filing or developed in the future. Reference is made to Rees & Liu, Base editing: precision chemistry on the genome and transcriptome of living cells, Nat. Rev. Genet. 2018;19(12):770-788; as well as.U.S. Patent Publication No. 2018/0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163; on October 30, 2018; U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019, as U.S. Patent No. 10,167,457; International Publication No. WO 2017/070633, published April 27, 2017; International Publication No. WO 2018/027078, published August 2, 2018;

International Application No PCT/US2018/056146, filed October 16, 2018, which published as Publication No. WO 2019/079347 on April 25, 2019; International Application No PCT/US2019/033848, filed May 23, 2019, which published as Publication No. WO

2019/226593 on November 28, 2019; U.S. Patent Publication No. 2015/0166980, published June 18, 2015; U.S. Patent No. 9,840,699, issued December 12, 2017; U.S. Patent No.

10,077,453, issued September 18, 2018; International Publication No. WO 2019/023680, published January 31, 2019; International Publication No. WO 2018/0176009, published September 27, 2018; International Application No. PCT/US2019/47996, filed August 23, 2019; International Application No. PCT/US2019/049793, filed September 5, 2019; U.S. Provisional Application No. 62/835,490, filed April 17, 2019; International Application No. PCT/US2019/61685, filed November 15, 2019; International Application No.

PCT/US2019/57956, filed October 24, 2019,, the contents of each of which are incorporated herein by reference in their entireties.

[0033] The term“Cas9” or“Cas9 nuclease” or“Cas9 domain” refers to a CRISPR associated protein 9, or functional fragment or variant thereof, and embraces any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. More broadly, a Cas9 protein, domain, or domain is a type of“nucleic acid programmable DNA binding protein (napDNAbp)”. The term Cas9 is not meant to be particularly limiting and may be referred to as a“Cas9 or equivalent.” Exemplary Cas9 proteins are described herein and also described in the art.

The present disclosure is unlimited with regard to the particular Cas9 that is employed in the base editors of the disclosure.

[0034] In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as“Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. Cas9 variants include functional fragments of Cas9. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9.

In some embodiments, the fragment is is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

[0035] As used herein, the term“dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment or variant thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a“dCas9 or equivalent.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference.

[0036] As used herein, the term“nCas9” or“Cas9 nickase” refers to a Cas9 or a functional fragment or variant thereof, which cleaves or nicks only one of the strands of a target cut site thereby introducing a nick in a double strand DNA molecule rather than creating a double strand break. This can be achieved by introducing appropriate mutations in a wild-type Cas9 which inactives one of the two endonuclease activities of the Cas9. Any suitable mutation which inactivates one Cas9 endonuclease activity but leaves the other intact is contemplated, such as one of D10A or H840A mutations in the wild-type Cas9 amino acid sequence (e.g., SEQ ID NO: 9) may be used to form the nCas9.

[0037] The term“continuous evolution,” as used herein, refers to an evolution procedure, (e.g., PACE) in which a population of nucleic acids is subjected to multiple rounds of (a) replication, (b) mutation, and (c) selection to produce a desired evolved product, for example, a nucleic acid encoding a protein with a desired activity, wherein the multiple rounds can be performed without investigator interaction and wherein the processes under (a)-(c) can be carried out simultaneously. Typically, the evolution procedure is carried out in vitro , for example, using cells in culture as host cells. In general, a continuous evolution process provided herein relies on a system in which a gene of interest is provided in a nucleic acid vector that undergoes a life-cycle including replication in a host cell and transfer to another host cell, wherein a critical component of the life-cycle is deactivated and reactivation of the component is dependent upon a desired mutation in the gene of interest. Reference is made to U.S Patent Publication No. 2013/0345064, which published on December 26, 2013 and issued as U.S. Patent No. 9,394,537 on July 19, 2016; and U.S Patent Publication No.

2016/0348096, which published on December 1, 2016 and issued as U.S. Patent No. 10, 179, 911, on January 15, 2019; and U.S Patent Publication No. 2017/0233708, which published August 17, 2017; and U.S Patent Publication No. 2017/0044520, which published on February 16, 2017, the contents of each of which are incorporated herein by reference in their entireties.

[0038] In some embodiments, the nucleic acid vector comprising the gene of interest is a phage, a viral vector, or naked DNA (e.g., a mobilization plasmid). In some embodiments, transfer of the gene of interest from cell to cell is via infection, transfection, transduction, conjugation, or uptake of naked DNA, and efficiency of cell-to-cell transfer (e.g., transfer rate) is dependent on the activity of a product encoded by the gene of interest. For example, in some embodiments, the nucleic acid vector is a phage harboring the gene of interest and the efficiency of phage transfer (via infection) is dependent on an activity of the gene of interest in that a protein required for the generation of phage particles (e.g., pill for M13 phage) is expressed in the host cells only in the presence of the desired activity of the gene of interest. In another example, the nucleic acid vector is a retroviral vector, for example, a lentiviral or vesicular stomatitis virus vector harboring the gene of interest, and the efficiency of viral transfer from cell to cell is dependent on the activity of the gene of interest in that a protein required for the generation of viral particles (e.g., an envelope protein, such as VSV- g) is expressed in the host cells only in the presence of the desired activity of the gene of interest. In another example, the nucleic acid vector is a DNA vector, for example, in the form of a mobilizable plasmid DNA, comprising the gene of interest, that is transferred between bacterial host cells via conjugation and the efficiency of conjugation-mediated transfer from cell to cell is dependent on an activity of the gene of interest in that a protein required for conjugation-mediated transfer (e.g., traA or traQ) is expressed in the host cells only in the presence of the desired activity of the gene of interest. Host cells contain F plasmid lacking one or both of those genes.

[0039] For example, some embodiments provide a continuous evolution system, in which a population of viral vectors comprising a gene of interest to be evolved replicates in a flow of host cells, e.g., a flow through a lagoon, wherein the viral vectors are deficient in a gene encoding a protein that is essential for the generation of infectious viral particles, and wherein that gene is comprised in the host cell under the control of a conditional promoter that can be activated by a gene product encoded by the gene of interest, or a mutated version thereof. In some embodiments, the activity of the conditional promoter depends on a desired function of a gene product encoded by the gene of interest. Viral vectors, in which the gene of interest has not acquired a mutation conferring the desired function, will not activate the conditional promoter, or only achieve minimal activation, while any mutation in the gene of interest that confers the desired mutation will result in activation of the conditional promoter. Since the conditional promoter controls an essential protein for the viral life cycle, activation of this promoter directly corresponds to an advantage in viral spread and replication for those vectors that have acquired an advantageous mutation.

[0040]“CRISPR” is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that have invaded the prokaryote. The snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively constitute, along with an array of CRISPR-associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (me), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently,

Cas9/crRNA/tracrRNA endonucleolytic ally cleaves linear or circular nucleic acid target complementary to the RNA. Specifically, the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gRNA”) may be engineered so as to incorporate embodiments of both the crRNA and tracrRNA into a single RNA species— the guide RNA. See, e.g., Jinek M., el al., Science 337:816-821(2012), the entire contents of which is herein incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti J.J., el al, Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001);“CRISPR RNA maturation by trans- encoded small RNA and host factor RNase III.” Deltcheva E., el al, Nature 471:602- 607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., el al, Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes, S. thermophiles, C. ulcerans, S. diphtheria, S.

syrphidicola, P. intermedia, S. taiwanense, S. iniae, B. baltica, P. torquis, S. thermophilus , L. innocua, C. jejuni and N. meningitidis. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0041] The term“effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a base editor may refer to the amount of the base editor that is sufficient to edit a target site of a nucleotide sequence, e.g., a genome. In some embodiments, an effective amount of a base editor provided herein, e.g., of a fusion protein comprising a nuclease-inactive Cas9 domain and a nucleobase modification domain (i.e., a thymine alkyltransferase domain) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. In some embodiments, an effective amount of a base editor provided herein may refer to the amount of the fusion protein sufficient to induce editing having the following characteristics: > 50% product purity, < 5% indels, and an editing window of 2-8 nucleotides. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a thymine alkyltransferase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the target cell or tissue (i.e., the cell or tissue to be edited), and on the agent being used.

[0042] The term“evolved base editor” or“evolved base editor variant” refers to a base editor formed as a result of mutagenizing a reference or starting-point base editor. The term refers to embodiments in which the nucleobase modification domain is evolved or a separate domain is evolved. Mutagenizing a reference or starting-point base editor may comprise mutagenizing a fusion protein comprising a thymine alkyltransferase by a continuous evolution method (e.g., PACE), wherein the evolved base editor has one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of the thymine alkyltransferase. Amino acid sequence variations may include one or more mutated residues within the amino acid sequence of a reference base editor, e.g., as a result of a change in the nucleotide sequence encoding the base editor that results in a change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing. The evolved base editor may include variants in one or more components or domains of the base editor (e.g., variants introduced into a Cas9 domain, a thymine alkyltransferase domain, an iDAR domain, or variants introduced into combinations of these domains).

[0043] The term“fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an“amino-terminal fusion protein” or a“carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0044] The term“host cell,” as used herein, refers to a cell that can host, replicate, and transfer a phage vector useful for a continuous evolution process as provided herein. In embodiments where the vector is a viral vector, a suitable host cell is a cell that can be infected by the viral vector, can replicate it, and can package it into viral particles that can infect fresh host cells. A cell can host a viral vector if it supports expression of genes of viral vector, replication of the viral genome, and/or the generation of viral particles. One criterion to determine whether a cell is a suitable host cell for a given viral vector is to determine whether the cell can support the viral life cycle of a wild-type viral genome that the viral vector is derived from. For example, if the viral vector is a modified M13 phage genome, as provided in some embodiments described herein, then a suitable host cell would be any cell that can support the wild-type M13 phage life cycle. Suitable host cells for viral vectors useful in continuous evolution processes are well known to those of skill in the art, and the disclosure is not limited in this respect. In some embodiments, the viral vector is a phage and the host cell is a bacterial cell. In some embodiments, the host cell is an E. coli cell. Suitable E. coli host strains will be apparent to those of skill in the art, and include, but are not limited to, New England Biolabs (NEB) Turbo, ToplOF’, DH12S, ER2738, ER2267, and XLl-Blue MRF’ . These strain names are art recognized and the genotype of these strains has been well characterized. It should be understood that the above strains are exemplary only and that the disclosure is not limited in this respect. The term“fresh,” as used herein interchangeably with the terms“non-infected” or“uninfected” in the context of host cells, refers to a host cell that has not been infected by a viral vector comprising a gene of interest as used in a continuous evolution process provided herein. A fresh host cell can, however, have been infected by a viral vector unrelated to the vector to be evolved or by a vector of the same or a similar type but not carrying the gene of interest.

[0045] In some embodiments, the host cell is a prokaryotic cell, for example, a bacterial cell. In some embodiments, the host cell is an E. coli cell. In some embodiments, the host cell is a eukaryotic cell, for example, a yeast cell, an insect cell, or a mammalian cell. The type of host cell, will, of course, depend on the viral vector employed, and suitable host cell/viral vector combinations will be readily apparent to those of skill in the art.

[0046] In some PACE embodiments, for example, in embodiments employing an M13 selection phage, the host cells are E. coli cells expressing the Fertility factor, also commonly referred to as the F factor, sex factor, or F-plasmid. The F-factor is a bacterial DNA sequence that allows a bacterium to produce a sex pilus necessary for conjugation and is essential for the infection of E. coli cells with certain phage, for example, with M13 phage. For example, in some embodiments, the host cells for M13-PACE are of the genotype F'proA⁺B⁺

A(lacIZY) zzf::Tnl0(TetR)/ endAl recAl galE15 galK16 nupG rpsF AlacIZYA araD139 A(ara,leu)7697 mcrA A(mrr-hsdRMS-mcrBC) proBA::pirl l6 l .

[0047] The term“linker,” as used herein, refers to a chemical group or a molecule linking two molecules or domains, e.g., nCas9 and a thymine alkyltransferase. In some

embodiments, a linker joins a dCas9 and modification domain (e.g., a thymine

alkyltransferase). Typically, the linker is positioned between, or flanked by, two groups, molecules, or other domains and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical domain. Chemical domains include, but are not limited to, disulfide, hydrazone, thiol and azo domains. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0048] The term“mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue; a deletion or insertion of one or more residues within a sequence; or a substitution of a residue within a sequence of a genome in a subject to be corrected. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include“loss-of- function” mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. There are some exceptions where a loss-of-function mutation is dominant, one example being

haploinsufficiency, where the organism is unable to tolerate the approximately 50% reduction in protein activity suffered by the heterozygote. This is the explanation for a few genetic diseases in humans, including Marfan syndrome which results from a mutation in the gene for the connective tissue protein called fibrillin. Mutations also embrace“gain-of-function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Alternatively the mutation could lead to overexpression of one or more genes involved in control of the cell cycle, thus leading to uncontrolled cell division and hence to cancer. Because of their nature, gain-of-function mutations are usually dominant. [0049] The terms“non-naturally occurring” or“engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides (e.g., Cas9 or thymine alkyltransferases) mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and/or as found in nature (e.g., an amino acid sequence not found in nature).

[0050] The term“nucleic acid,” as used herein, refers to RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms“nucleic acid,”“DNA,”“RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids may be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids may comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0051] The term“nucleic acid programmable D/RNA binding protein (napR/DNAbp)” refers to any protein that may associate (e.g., form a complex) with one or more nucleic acid molecules (i.e., which may broadly be referred to as a“napR/DNAbp-programming nucleic acid molecule” and includes, for example, guide RNA in the case of Cas systems) which direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the protein to bind to the nucleotide sequence at the specific target site. This term napR/DNAbp embraces CRISPR Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or modified), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), C2c3 (a type V CRISPR-Cas system), dCas9, GeoCas9, CjCas9, Casl2a, Casl2b, Casl2c, Casl2d, Casl2g, Casl2h, Casl2i, Casl3b, Casl3c, Casl3d, Casl4, Csn2, Argonaute (Ago), and nCas9. The term also embraces Cas homologs and variants such as an xCas9, an SpCas9-NG, an LbCasl2a, an AsCasl2a, a Cas9-KKH, a circularly permuted Cas9, a SmacCas9, a Spy-macCas9. Further Cas-equivalents are described in Makarova el al.,“C2c2 is a single-component

programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference. However, the nucleic acid programmable DNA binding protein (napDNAbp) that may be used in connection with this disclosure are not limited to CRISPR-Cas systems. The disclosure embraces any such programmable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo) which may also be used for DNA-guided genome editing.

[0052] In some embodiments, napR/DNAbp is a RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though“gRNA” is used interchangeabley to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 (or equivalent) complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is homologous to a tracrRNA as depicted in Figure IE of Jinek et al, Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Patent No. 9,340,799, entitled“mRNA-Sensing Switchable gRNAs,” and International Patent Application No. PCT/US2014/054247, filed September 6, 2013, published as WO 2015/035136 and entitled“Delivery System For Functional Nucleases,” the entire contents of each are herein incorporated by reference. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an“extended gRNA.” For example, an extended gRNA will, e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA- programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example Cas9 (Csnl) from Streptococcus pyogenes (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes” Ferretti J.J. et al.., Proc. Natl. Acad. Sci. U.S.A. 98:4658- 4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al, Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al, Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference.

[0053] The napR/DNAbp nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using napR/DNAbp nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J.E. et al., Genome engineering in

Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acid Res. (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature

Biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0054] The term“napR/DNAbp-programming nucleic acid molecule” or equivalently“guide sequence” refers the one or more nucleic acid molecules which associate with and direct or otherwise program a napR/DNAbp protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the napR/DNAbp protein to bind to the nucleotide sequence at the specific target site. A non limiting example is a guide RNA of a Cas protein of a CRISPR-Cas genome editing system.

[0055] A nuclear localization signal or sequence (NLS) is an amino acid sequence that tags, designates, or otherwise marks a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus. Thus, a single nuclear localization signal can direct the entity with which it is associated to the nucleus of a cell. Such sequences can be of any size and composition, for example more than 25, 25, 15, 12, 10, 8, 7, 6, 5 or 4 amino acids, but will preferably comprise at least a four to eight amino acid sequence known to function as a nuclear localization signal (NLS).

[0056] The term, as used herein,“nucleobase modification domain” or“modification domain” embraces any protein, enzyme, or polypeptide (or functional fragment thereof) which is capable of modifying a DNA or RNA molecule. Nucleobase modification domains may be naturally occurring, or may be engineered. For example, a nucleobase modification domain can include one or more DNA repair enzymes, for example, and an enzyme or protein involved in base excision repair (BER), nucleotide excision repair (NER), homology- dependnent recombinational repair (HR), non-homologous end-joining repair (NHEJ), microhomology end-joining repair (MMEJ), mismatch repair (MMR), direct reversal repair, or other known DNA repair pathway. A nucleobase modification domain can have one or more types of enzymatic activities, including, but not limited to, endonuclease activity, polymerase activity, ligase activity, replication activity, and proofreading activity.

Nucleobase modification domains can also include DNA or RNA-modifying enzymes and/or mutagenic enzymes, such as DNA alkyltransferases (i.e., thymine alkyl transferases), which covalently modify nucleobases leading in some cases to mutagenic corrections by way of normal cellular DNA repair and replication processes. Exemplary nucleobase modification domains include, but are not limited to, a thymine alkyltransferase (e.g., RsmE), a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.

[0057] As used herein, the terms“oligonucleotide” and“polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three

nucleotides). [0058] The term“phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors. The general concept of PACE technology has been described, for example, in International PCT Application,

PCT/US 2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application, PCT/US2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Application, U.S. Patent No.

9,023,594, issued May 5, 2015, International PCT Application, PCT/US2015/012022, filed January 20, 2015, published as WO 2015/134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued January 15, 2019; International PCT Application, PCT/US2016/027795, filed April 15, 2016, published as WO 2016/168631, on October 20, 2016, and International Patent Publication WO 2019/023680, published January 31, 2019, the entire contents of each of which are incorporated herein by reference.

[0059] The term“phage-assisted non-continuous evolution (PANCE),” as used herein, refers to non-continuous evolution that employs phage as viral vectors. The general concept of PANCE technology has been described, for example, in Suzuki T. et al, Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13(12): 1261-1266 (2017), incorporated herein by reference in its entirety. Briefly, PANCE is a simplified technique for rapid in vivo directed evolution using serial flask transfers of evolving‘selection phage’ (SP), which contain a gene of interest to be evolved, across fresh E. coli host cells, thereby allowing genes inside the host E. coli to be held constant while genes contained in the SP continuously evolve. Following phage growth, an aliquot of infected cells is used to transfect a subsequent flask containing host E. coli. This process is continued until the desired phenotype is evolved, for as many transfers as required. Serial flask transfers have long served as a widely-accessible approach for laboratory evolution of microbes, and, more recently, analogous approaches have been developed for bacteriophage evolution. The PANCE system features lower stringency than the PACE system.

[0060] The term“promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule“inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant disclosure, which is not limited in this respect. In various embodiments, the disclosure provides vectors with appropriate promoters for driving expression of the nucleic acid sequences encoding the base editor fusion proteins (or one or more individual components thereof).

[0061] The term“phage,” as used herein interchangeably with the term“bacteriophage,” refers to a virus that infects bacterial cells. Typically, phages consist of an outer protein capsid enclosing genetic material. The genetic material may be ssRNA, dsRNA, ssDNA, or dsDNA, in either linear or circular form. Phages and phage vectors are well known to those of skill in the art and non-limiting examples of phages that are useful for carrying out the methods provided herein are l, T2, T4, T7, T12, R17, M13, MS2, G4, PI, P2, P4, Phi X174, N4, F6, and F29. In certain embodiments, the phage utilized in the present disclosure is M13. Additional suitable phages and host cells will be apparent to those of skill in the art and the disclosure is not limited in this aspect. For an exemplary description of additional suitable phages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze:

Bacteriophages: Biology and Applications . CRC Press; 1^st edition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology ) Humana Press; 1^st edition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Embodiments (Methods in Molecular Biology) Humana Press; 1^st edition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable phages and host cells as well as methods and protocols for isolation, culture, and manipulation of such phages). The terms“protein,” “peptide,” and“polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, engineered, or synthetic, or any combination thereof. The term“fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an“amino- terminal fusion protein” or a“carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a recombinase. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0062] The term“recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

[0063] The term“subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

[0064] The term“target site” refers to a sequence within a nucleic acid molecule that is edited by a base editor (e.g., a dCas9-thymine alkyltransferase fusion protein provided herein). The target site further refers to the sequence within a nucleic acid molecule to which a complex of the base editor and gRNA binds.

[0065] The term“vector,” as used herein, may refer to a nucleic acid that has been modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Alternatively, the term“vector” as used herein may refer to a nucleic acid that has been modified to encode the base editor. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids.

[0066] The term“viral particle,” as used herein, refers to a viral genome, for example, a DNA or RNA genome, that is associated with a coat of a viral protein or proteins, and, in some cases, with an envelope of lipids. For example, a phage particle comprises a phage genome packaged into a protein encoded by the wild type phage genome.

[0067] The term“viral vector,” as used herein, refers to a nucleic acid comprising a viral genome that, when introduced into a suitable host cell, can be replicated and packaged into viral particles able to transfer the viral genome into another host cell. The term“viral vector” extends to vectors comprising truncated or partial viral genomes. For example, in some embodiments, a viral vector is provided that lacks a gene encoding a protein essential for the generation of infectious viral particles. In suitable host cells, for example, host cells comprising the lacking gene under the control of a conditional promoter, however, such truncated viral vectors can replicate and generate viral particles able to transfer the truncated viral genome into another host cell. In some embodiments, the viral vector is an adeno- associated virus (AAV) vector.

[0068] The terms“treatment,”“treat,” and“treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease, disorder, or condition, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,”“treat,” and“treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease, disorder, or condition, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be

administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their prevention or recurrence.

[0069] As used herein, the term“variant” refers to a protein having characteristics that deviate from what occurs in nature or a base sequence and that retains at least one functional property, i.e., binding, interaction, or enzymatic activity, and/or therapeutic property thereof. A“variant” is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the wild type protein. For instance, a variant of Cas9 may comprise a Cas9 that has one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. As another example, a variant of a deaminase may comprise a deaminase that has one or more changes in amino acid residues as compared to a wild type deaminase amino acid sequence, e.g. following ancestral sequence reconstruction of the deaminase. These changes include chemical modifications, substitutions of different amino acid residues, truncations, covalent additions (e.g., addition of a tag), and any other changes. This term also embraces fragments of a wild type protein.

[0070] The level or degree of which the property is retained may be reduced relative to the wild type protein but is typically the same or similar in kind. Generally, variants are overall very similar, and in many regions, identical to the amino acid sequence of the protein described herein. A skilled artisan will appreciate how to make and use variants that maintain all, or at least some, of a functional ability or property.

[0071] The variant proteins may comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, identical to, for example, the amino acid sequence of a wild-type protein, or any protein provided herein. Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding a protein such as a napDNAbp under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6x Sodium chloride/S odium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2. times. SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (e.g. hybridization to filter bound DNA in 6x sodium chloride/S odium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in O.lxSSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et ah, eds., 1989 Current Protocol in Molecular Biology , Green Publishing Associates, Inc., and John Wiley & Sons Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3).

[0072] By a polypeptide having an amino acid sequence at least, for example, 95%

“identical” to a query amino acid sequence, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0073] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the amino acid sequence of a protein such as a napDNAbp, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. {Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k- tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

[0074] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the reference sequence.

[0075] As used herein the term“wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0076] Disclosed herein are novel thymine-to-adenine or“TABE” transversion base editors which comprise a napDNAbp (e.g., a dCas9 domain) fused to a nucleobase modification domain, i.e. a thymine alkyltransferase domain. The TABE transversion base editors are capable of converting a T:A nucleobase pair to an A:T nucleobase pair in a target nucleotide sequence of interest, e.g., the genome of a cell.

[0077] The present inventors have developed novel transversion base editors, and in particular a novel base editor that installs a T-to-A transversion in a targeted manner, through a thymine alkylation reaction. The disclosed TABE base editors comprise an alkyltransferase domain, or variant thereof, that catalyzes the conversion of a target thymine to an adenine via an alkylation reaction.

[0078] The disclosed base editors also comprise TABE transversion base editors that comprise an alkyltransferase domain, or variant thereof, that catalyzes the conversion of a target thymine to an adenine via an alkylation reaction, wherein the base-paired adenine of the non-edited strand is subsequently converted to a thymine by the concerted action of the cell’s mismatch repair factors.

[0079] The present disclosure provides for the enzyme-catalyzed addition of an alkyl group, e.g. a methyl or carboxymethyl group, to a targeted T in a nucleic acid of interest is induced, resulting in N3-methyl thymine or N3 -carboxymethyl thymine formation, respectively. In another embodiment, the enzyme-catalyzed addition of a 3-amino-3-carboxypropyl group to a targeted T in a nucleic acid of interest is induced, resulting in the formation of an N3-3- amino-3-carboxypropyl thymine. Each of N3-methyl thymine, N3-carboxymethyl thymine, and N3-3-amino-3-carboxypropyl thymine disrupts the hydrogen bonding interactions with the base-paired adenine of the unmutated strand. Without wishing to be bound by any particular theory, the cell’s replication machinery interprets the alkylated thymine as an adenine and converts the mismatched adenine of the unmutated strand to a thymine. During a subsequent round of replication or mismatch repair, the alkylated thymine is converted to an adenine. A desired T-to-A transversion is thus achieved. Thymine alkylation is achieved by the targeted use of a fusion protein comprising a Cas9 (e.g., a catalytically dead Cas9

(“dCas9”) or Cas9 nickase (“nCas9”)) domain, a thymine alkyltransferase domain, and optionally a linker connecting these two domains (see FIGs. 1A, 2A).

[0080] The disclosure provides a base editor fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) a thymine alkyltransferase. In various embodiments of the base editor fusion proteins, the thymine alkyltransferase is a wild-type thymine alkyltransferase. In certain embodiments, the thymine alkyltransferase is a wild-type RsmE, or a variant thereof, that methylates a thymine in a nucleic acid. In certain embodiments, the RsmE is an Escherichia coli RsmE, or a variant thereof. In certain embodiments, the RsmE variant has been evolved to accept a modified 5-adcnosyl- methionine (SAM) cofactor, e.g. a SAM cofactor modified to include a carboxymethyl domain on the S⁺ center, so as to yield an N3 -carboxymethyl thymine product.

[0081] The thymine alkyltransferase domains of the disclosed base editors may comprise variants of wild-type alkyltransferase enzymes. These variants may comprise an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the wild type enzyme. In some embodiments, the thymine alkyltransferase domains may comprise an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more than 30 amino acids that differ relative to the amino acid sequence of the wild type enzyme. These differences may comprise nucleotides that have been inserted, deleted, or substituted relative to the amino acid sequence of the wild type enzyme. In some embodiments, the thymine alkyltransferase domains contain stretches of about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, or more than 500 consecutive amino acids in common with the wild type enzyme. In some embodiments, the thymine alkyltransferase domains comprise truncations at the N-terminus or C-terminus relative to the wild-type enzyme. In some embodiments, the thymine alkyltransferase domains comprise truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more than 30 amino acids at the N-terminus or C-terminus relative to the wild-type or base sequence.

[0082] In certain embodiments, the thymine alkyltransferase is a methyltransferase that methylates a thymine to N3-methylthymine. In various embodiments, the alkyltransferase comprises a RsmE, or a variant thereof, that methylates thymine in a nucleic acid. In other embodiments, the alkyltransferase comprises a Saccharomyces cerevisiae Bmt5,

Saccharomyces cerevisiae Bmt6, or a variant thereof. In other embodiments, the

alkyltransferase is a DNA methyltransferase (DNMT), such as DNMT1, DNMT3a,

DNMT3b, or DNMT3L. In some embodiments, the alkyltransferase comprises a human DNMT3a, a human DNMT3b, or a human DNMT3L.

[0083] In other embodiments, the thymine alklytransferase comprises a CpG

Methyltransferase (M.SssI), e.g. an M.SssI from Spiroplasma monobiae. In other

embodiments, the thymine alkyltransferase comprises an Hhal methyltransferase, e.g. an Hhal methyltransferase from Haemophilus parahaemolyticus .

[0084] In other embodiments, the thymine alkyltransferase is a carboxymethyltransferase that attaches a carboxymethyl group to a thymine to form N3-carboxymethylthymine. In certain embodiments, the alkyltransferase has been evolved to accept as its substrate a modified S- adenosyl-methionine (SAM) cofactor. In certain embodiments, the alkyltransferase has been evolved to accept as its substrate a SAM cofactor modified to include a carboxymethyl domain on the S⁺ center, so as to yield an N3 -carboxymethyl thymine product.

[0085] In other embodiments, the thymine alkyltransferase is an amino-carboxy- propyltransferase, which attaches a 3-amino-3-carboxypropyl group to a thymine to form N3- 3-amino-3-carboxyproylthymine. In certain embodiments, the alkyltransferase is a wild-type Tsr3, or a variant thereof, which attaches a 3-amino-3-carboxypropyl group to a thymine in a nucleic acid. In certain embodiments, the Tsr3 is a human Tsr3, or a variant thereof. In other embodiments, the Tsr3 is a Saccharomyces cerevisiae Tsr3, a Vulcaniseta distributa Tsr3, a Sulfolobus solfataricus Tsr3, or a variant thereof.

[0086] The present disclosure provides for T:A to A:T transversion base editors which satisfy a need in the art for the installation of targeted transversions in a target nucleotide sequence, e.g., a genome. In particular, the present disclosure provides T:A to A:T base editors (e.g., fusion proteins comprising a dCas9 domain and a thymine alkyltransferase domain) which satisfy a need in the art for effecting targeted transversions, particularly T:A to A:T trans versions. In addition, the disclosure provides compositions comprising the transversion base editors as described herein, e.g., fusion proteins comprising a dCas9 domain and a thymine alkyltransferase domain. In addition, the present disclosure provides for nucleic acid molecules encoding and/or expressing the transversion base editors as described herein, as well as expression vectors and constructs for expressing the transversion base editors described herein, host cells comprising said nucleic acid molecules and expression vectors, and compositions for delivering and/or administering nucleic acid-based embodiments described herein.

[0087] Still further, the present disclosure provides for methods of making the transversion base editors, as well as methods of using the transversion base editors or nucleic acid molecules encoding the transversion base editors in applications including editing a nucleic acid molecule, e.g., a genome. In certain embodiments, methods of engineering the transversion base editors provided herein is a phage-assisted continuous evolution (PACE) system or non-continuous system (e.g., PANCE) which may be utilized to evolve one or more components of a base editor (e.g., a Cas9 domain or a thymine alkyltransferase domain). In certain embodiments, following the successful evolution of the one or more components of the transversion base editor, methods of making the base editors comprise recombinant protein expression methodologies known to one of ordinary skill in the art.

[0088] The specification also provides methods for editing a target nucleic acid molecule, e.g., a single nucleobase within a genome, with a base editing system described herein (e.g., in the form of an evolved base editor as described herein, or a vector or construct encoding same). Such methods involve transducing (e.g., via transfection) cells with a plurality of complexes each comprising a fusion protein (e.g., a fusion protein comprising a Cas9 nickase (nCas9) domain and an thymine alkyltransferase domain) and a gRNA molecule. In some embodiments, the gRNA is bound to the napDNAbp domain (e.g., nCas9 domain) of the fusion protein. In some embodiments, each gRNA comprises a guide sequence of at least 10 contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that is complementary to a target sequence. In certain embodiments, the methods involve the transfection of nucleic acid constructs (e.g., plasmids) that each (or together) encode the components of a complex of fusion protein and gRNA molecule. [0089] In certain embodiments of the disclosed methods, a nucleic acid construct that encodes the fusion protein is transfected into the cell separately from the plasmid that encodes the gRNA molecule. In certain embodiments, these components are encoded on a single construct and transfected together.

[0090] In other embodiments, the methods disclosed herein involve the introduction into cells of a complex comprising a fusion protein and gRNA molecule that has been expressed and cloned outside of these cells.

[0091] It should be appreciated that any fusion protein, e.g., any of the fusion proteins provided herein, may be introduced into the cell in any suitable way, either stably or transiently. In some embodiments, a fusion protein may be transfected into the cell. In some embodiments, the cell may be transduced or transfected with a nucleic acid construct that encodes a fusion protein. For example, e a cell may be transduced (e.g., with a virus encoding a fusion protein), or transfected (e.g., with a plasmid encoding a fusion protein) with a nucleic acid that encodes a fusion protein, or the translated fusion protein. Such transduction may be a stable or transient transduction. In some embodiments, cells expressing a fusion protein or containing a fusion protein may be transduced or transfected with one or more gRNA molecules, for example when the fusion protein comprises a Cas9 (e.g., nCas9) domain. In some embodiments, a plasmid expressing a fusion protein may be introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction or other methods known to those of skill in the art.

[0092] In certain embodiments, the methods described herein result in a cutting (or nicking) one strand of the double- stranded DNA, for example, the strand that includes the adenine (A) of the target A:T nucleobase pair opposite the strand containing the target thymine (T) that is being alkylated. This nicking result serves to direct mismatch repair machinery to the non- edited strand, ensuring that the chemically modified nucleobase is not interpreted as a lesion by the machinery. This nick may be created by the use of an nCas9.

[0093] The specification also provides methods for efficiently editing a target nucleic acid molecule, e.g., a single nucleobase of a genome, with a base editing system described herein (e.g., in the form of an base editor as described herein or a vector or construct encoding same), thereby installing a transversion edit. Still further, the disclosure provides therapeutic methods for treating a genetic disease and/or for altering or changing a genetic trait or condition by contacting a target nucleic acid molecule, e.g., a target nucleic acid molecule in the genome of an organism, with a base editing system (e.g., in the form of an base editor protein or a vector encoding same) and conducting base editing to treat the genetic disease and/or change the genetic trait (e.g., eye color).

[0094] In various embodiments, a method is provided for editing a nucleobase pair of a double-stranded DNA sequence, the method comprising: (i) contacting a double- stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid, wherein the double- stranded DNA comprises a target T:A nucleobase pair; and (ii) alkylating the thymine (T) of the T:A nucleobase pair to an alkylated thymine (e.g. N3-methyl thymine, N3- carboxymethyl thymine, or N3-3-amino-3-carboxypropyl thymine). In various embodiments, the alkylated thymine is subsequently replaced with an adenine (A), thereby generating a T to A change.

[0095] In other embodiments, the present disclosure provides a complex comprising the base editor fusion proteins described herein and an RNA bound to the napDNAbp of the fusion protein, such as a guide RNA (gRNA), e.g. a single guide RNA.

[0096] The target nucleotide sequence may comprise a target sequence (e.g., a point mutation) associated with a disease, disorder, or condition, such as sickle cell anemia,

Fanconi anemia, ectodermal dysplasia skin fragility syndrome, lattice corneal dystrophy Type III, or Noonan syndrome. The target sequence may comprise an A to T point mutation associated with a disease, disorder, or condition, and wherein the alkylation of the mutant T base results in mismatch repair- mediated correction to a sequence that is not associated with a disease, or disorder, or condition. The target sequence may otherwise comprise a T to A point mutation associated with a disease, or disorder, or condition, wherein the alkylation of the T base opposite the mutant A base results in mismatch repair-mediated correction to a sequence that is not associated with the disease, or disorder, or condition. The target sequence may encode a protein, and where the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to a wild-type codon.

The target sequence may also be at a splice site, and the point mutation results in a change in the splicing of an mRNA transcript as compared to a wild-type transcript. In addition, the target may be at a non-coding sequence of a gene, such as a promoter, and the point mutation results in increased or decreased expression of the gene.

[0097] Exemplary target genes include HBB, in which an A to T point mutation at residue 334 results in a sickle cell anemia phenotype; and FANCC, in which an A to T point mutation at residue 456 results in a Fanconi anemia phenotype. Additional target genes include TGFBI (associated with lattice comeal dystrophy type III), PKP1 (associated with ectodermal dysplasiaskin fragility syndrome), KRAS and SOS1 (both associated with Noonan syndrome), for which the disease phenotype is frequently caused by T:A to A:T point mutations.

[0098] In another aspect, the specification discloses a pharmaceutical composition comprising any one of the presently disclosed base editor fusion proteins. In one aspect, the specification discloses a pharmaceutical composition comprising any one of the presently disclosed complexes of fusion proteins and gRNA. In one aspect, the specification discloses a pharmaceutical composition comprising polynucleotides encoding the fusion proteins disclosed herein and polynucleotides encoding a gRNA, or polynucleotides encoding both.

[0099] In another aspect, the specification discloses a pharmaceutical composition comprising any one of the presently disclosed vectors. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition further comprises a lipid and/or polymer. In certain embodiments, the lipid and/or polymer is cationic. The preparation of such lipid particles is well known. See, e.g. U.S. Patent Nos. 4,880,635; 4,906,477;

4,911,928; 4,917,951; 4,920,016; 4,921,757; and 9,737,604, each of which is incorporated herein by reference.

[0100] In various embodiments, the present disclosure provides T-to-A (or A-to-T) transversion base editor fusion proteins comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) a nucleobase modification domain capable of facilitating the conversion of a T:A nucleobase pair to an A:T nucleobase pair in a target nucleotide sequence, e.g., a genome.

[0101] In various embodiments, the nucleobase modification domain is a thymine

alkyltransferase, which enzymatically converts a thymine nucleobase of a T:A nucleobase pair to an an alkylated thymine, which then is subsequently processed by the cell’s DNA repair and replication machinery to an adenine, ultimately converting the T:A nucleobase pair to an A:T nucleobase pair.

[0102] The various domains of the transversion fusion proteins described herein (e.g., the Cas9 domain or the nucleobase modification domains) may be obtained as a result of mutagenizing a reference or starting-point base editor (or a component or domain thereof) by an evolution or modification strategy. Such strategies include a directed evolution process, e.g., a continuous evolution method (e.g., PACE) or a non-continuous evolution method (e.g., PANCE or other discrete plate-based selections). In various embodiments, the disclosure provides a base editor that has one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of the reference or starting-point base editor. The base editor may include variants in one or more components or domains of the base editor (e.g., variants introduced into a Cas9 domain, a thymine alkyltransferase domain, an inhibitor of DNA alkylation repair (iDAR) domain, or variants introduced into

combinations of these domains). For example, the nucleobase modification domain may be evolved from a reference protein that is an RNA modifying enzyme (e.g., uridine rRNA methyltransferases) and evolved using PACE, PANCE, or other plate-based evolution methods to obtain a DNA modifying version of the nucleobase modification domain, which can then be used in the fusion proteins described herein.

I. napDNAbp domains

[0103] The base editors described herein comprise a nucleic acid programmable DNA binding (napDNAbp) domain. The napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp domain to localize and bind to a complementary sequence of the target strand. Binding of the napDNAbp domain to a complementary sequence enables the nucleobase modification domain of the base editor to access and enzymatically deaminate a target adenine base in the target strand.

[0104] The napDNAbp can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (me) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3- aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5'

exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek et al, Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. [0105] Without wishing to be bound by any particular theory, the binding mechanism of a napDNAbp - guide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guideRNA protospacer then hybridizes to the“target strand.” This displaces a“non-target strand” that is

complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which cuts the DNA leaving various types of lesions ( e.g ., a nick in one strand of the DNA). For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a“double- stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is“nicked” on one strand.

[0106] The below description of various napDNAbps which can be used in connection with the disclosed nucleobase modification domains is not meant to be limiting in any way. The base editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein— including any naturally occurring variant, mutant, or otherwise engineered version of Cas9— that is known or which can be made or evolved through a directed evolution or otherwise mutagenic process. In various embodiments, the napDNAbp has a nickase activity, i.e., only cleave one strand of the target DNA sequence. In other

embodiments, the napDNAbp has an inactive nuclease, e.g., are“dead” proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid sequence (e.g., the circular permutant forms). The base editors described herein may also comprise Cas9 equivalents, including Casl2a/Cpfl and Casl2b proteins. The napDNAbps used herein (e.g., an SpCas9 or SpCas9 variant) may also may also contain various modifications that alter/enhance their PAM specifities. The disclosure contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a reference SpCas9 canonical sequence (set forth in SEQ ID NO: 9), a reference SaCas9 canonical sequence (set forth in SEQ ID NO: 86) or a reference Cas9 equivalent (e.g., Casl2a/Cpfl). [0107] In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence ansacs9d/or within the complement of the target sequence. In some embodiments, the napDNAbp directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents.

[0108] As used herein, the term“Cas protein” refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al.,“C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference.

[0109] The term“Cas9” or“Cas9 domain” embraces any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a“Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular napDNAbp that is employed in the base editors of the disclosure. [0110] Additional Cas9 sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et ah, J J., McShan W.M., Ajdic D.J., Savic D J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara L, Hauer M., Doudna J.A., Charpentier E. Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference), and also provided below.

[0111] Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The base editors of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.

A. Wild type canonical SpCas9

[0112] In one embodiment, the base editor constructs described herein may comprise the “canonical SpCas9” nuclease from S. pyogenes, which has been widely used as a tool for genome engineering. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. As used herein, the canonical SpCas9 protein refers to the wild type protein from

Streptococcus pyogenes having the following amino acid sequence:

[0113] The base editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above. These variants may include SpCas9 variants containing one or more mutations, including any known mutation reported with the SwissProt Accession No. Q99ZW2 entry, which include:

[0114] Other wild type SpCas9 sequences that may be used in the present disclosure, include:

[0115] The base editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least

99% sequence identity thereto.

B. Wild type Cas9 orthologs

[0116] In other embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species. For example, the following Cas9 orthologs can be used in connection with the base editor constructs described in this disclosure. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the disclosed base editors.

[0117] The base editors described herein may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0118] The napDNAbp may include any suitable homologs and/or orthologs or naturally occurring enzymes, such as Cas9. Cas9 homologs and/or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus . Preferably, the Cas moiety is configured (e.g, mutagenized, recombinantly engineered, or otherwise obtained from nature) as a nickase, i.e., capable of cleaving only a single strand of the target doubpdditional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 3. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the Cas9 orthologs in the above tables.

C. Dead napDNAbp variants

[0119] In some embodiments, the disclosed base editors may comprise a catalytically inactive, or“dead,” napDNAbp domain. Exemplary catalytically inactive domains in the disclosed base editors are dead S. pyogenes Cas9 (dSpCas9) and S. pyogenes Cas9 nickase (SpCas9n).

[0120] In certain embodiments, the base editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactivate both nuclease domains of SpCas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation may be due to one or mutations that result in one or more substitutions and/or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0121] In certain embodiments, the base editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactivate both nuclease domains of SpCas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The D10A and N580A mutations in the wild-type S. aureus Cas9 amino acid sequence may be used to form a dSaCas9. Accordingly, in some embodiments, the napDNAbp domain of the base editors provided herein comprises a dSaCas9 that has D10A and N580A mutations relative to the wild-type SaCas9 sequence (SEQ ID NO: 85.

[0122] As used herein, the term“dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a“dCas9 or equivalent.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference.

[0123] In other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided which may result in the full or partial inactivate of the endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations, by way of example, include other amino acid substitutions at DIO and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvCl subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1)) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to NCBI Reference Sequence: NC_017053.1. In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1) are provided having amino acid sequences which are shorter, or longer than NC_017053.1 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

[0124] In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises a dead S. pyogenes Cas9 (dSpCas9). In some embodiments, the napDNAbp domain of any of the disclosed based editors is comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 99. In some

embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 99.

[0125] In some embodiments, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and may have the following sequence, which comprises a D10A and an H810A substitutions (underlined and bolded), or a variant of SEQ ID NO: 99 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto:

D. napDNAbp nickase variants

[0126] In some embodiments, the disclosed base editors may comprise a napDNAbp domain that comprises a nickase. In some embodments, the base editors described herein comprise a Cas9 nickase. The term“Cas9 nickase” of“nCas9” refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain. The wild type Cas9 (e.g., the canonical SpCas9) comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762, have been reported as loss-of-function mutations of the RuvC nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et ah,“Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain could include D10X, H983X, D986X, or E762X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be D10A, of H983A, or D986A, or E762A, or a combination thereof.

[0127] In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises an S. pyogenes Cas9 nickase (SpCas9n). In some embodiments, the napDNAbp domain of any of the disclosed based editors is comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 105 or 111. In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 111.

[0128] In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises an S. aureus Cas9 nickase (SaCas9n). In some embodiments, the napDNAbp domain of any of the disclosed based editors is comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 109 In some

embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 109.

[0129] In various embodiments, the Cas9 nickase can having a mutation in the RuvC nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0130] In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain which inactivates the HNH nuclease activity. For example, mutations in histidine (H) 840 or asparagine (R) 863 have been reported as loss-of-function mutations of the HNH nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu el al,“Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain could include H840X and R863X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be H840A or R863A or a combination thereof.

[0131] In various embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0132] In some embodiments, the N-terminal methionine is removed from a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus Cas9 nickases include the following sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

E. Other Cas9 variants

[0133] The napDNAbp domains used in the base editors described herein may also include other Cas9 variants that area at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SEQ ID NO: 9).

[0134] In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0135] In various embodiments, the base editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.

F. Other Cas9 equivalents

[0136] In some embodiments, the base editors described herein can include any Cas9 equivalent. As used herein, the term“Cas9 equivalent” is a broad term that encompasses any napDNAbp protein that serves the same function as Cas9 in the present base editors despite that its amino acid primary sequence and/or its three-dimensional structure may be different and/or unrelated from an evolutionary standpoint. Thus, while Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are evolutionarily related, the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and/or three dimensional structure. The base editors described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution.

[0137] For example, CasX is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution. Thus, the CasX protein described in Liu et al.,“CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature , 2019, Vol.566: 218-223, is contemplated to be used with the base editors described herein.

In addition, any variant or modification of CasX is conceivable and within the scope of the present disclosure.

[0138] Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.

[0139] In some embodiments, Cas9 equivalents may refer to CasX or CasY, which have been described in, for example, Burstein et ah,“New CRISPR-Cas systems from

uncultivated microbes.” Cell Res. 2017 Feb 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little- studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. Also see Liu et ah, “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature , 2019, Vol.566: 218-223. Any of these Cas9 equivalents are contemplated.

[0140] In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.

[0141] In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 ( e.g dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, Argonaute, Casl2a, and Casl2b. One example of a nucleic acid programmable DNA- binding protein that has different PAM specificity than Cas9 is Clustered Regularly

Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpfl). Similar to Cas9, Cpfl is also a class 2 CRISPR effector. It has been shown that Cpfl mediates robust DNA interference with features distinct from Cas9. Cpfl is a single RNA-guided

endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf 1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpfl proteins are known in the art and have been described previously, for example Yamano et al,“Crystal structure of Cpfl in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. The state of the art may also now refer to Cpfl enzymes as Cas 12a.

[0142] In still other embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to Casl2a, Casl2b, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (sometimes referred to as Csnl and Csxl2), CaslO, Csyl, Csy2,

Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, and preferably comprising a nickase mutation ( e.g ., a mutation corresponding to the D10A mutation of the wild type SpCas9 polypeptide of SEQ ID NO: 9).

[0143] In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Cpfl, a CasX, a CasY, a C2cl, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Casl2a, a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago), a Cas9-KKH, a SmacCas9, a Spy-macCas9, an SpCas9-VRQR, an SpCas9-NRRH, an SpaCas9-NRTH, an SpCas9-NRCH, or a variant thereof.

[0144] In certain embodiments, the base editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence. In some embodiments, the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. The term“small-sized Cas9 variant”, as used herein, refers to any Cas9 variant— naturally occurring, engineered, or otherwise— that is less than at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acids, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein.

[0145] In various embodiments, the base editors disclosed herein may comprise one of the small-sized Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference small-sized Cas9 protein. Exemplary small-sized Cas9 variants include, but are not limited to, SaCas9 and LbCasl2a. [0146] In some embodiments, the base editors described herein may also comprise

Casl2a/Cpfl (dCpfl) variants that may be used as a guide nucleotide sequence- programmable DNA-binding protein domain. The Casl2a/Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alpha-helical recognition lobe of Cas9. It was shown in Zetsche el al, Cell , 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpfl is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpfl nuclease activity.

[0147] Additional exemplary Cas9 equivalent protein sequences can include the following:

Description Sequence

G. napDNAbps that recognize non-canonical PAM sequences

[0148] In some embodiments, the napDNAbp is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5' phosphorylated ssDNA of ~24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a

protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al, Nat Biotechnol., 2016 Jul;34(7):768-73. PubMed PMID: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference.

[0149] In some embodiments, the disclosure provides napDNAbp domains that comprise SpCas9 variants that recognize and work best with NRRH, NRCH, and NRTH PAMs. See PCT Application No. PCT/US2019/47996, incorporated by reference herein. In some embodiments, the disclosed base editors comprise a napDNAbp domain selected from SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH.

[0150] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-NRRH. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-NRRH. The SpCas9-NRRH has an amino acid sequence as presented in SEQ ID NO: 134 (underligned residues are mutated relative to SpCas9, as set forth in SEQ ID NO: 9):

MDKKY S IGLDIGTNS VGWAVITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLFDS GET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIF GNIVDE VAYHEKYPTIYHLRKKLVDS TD KADLRLIYLALAHMIKFRGHFLIEGDLN PDNS D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLI AQLPGEK KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYK EIFFDQS KN G YAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD N GIIPHQIHLGELH AILRRQGDFYPFLKDNREKIEKILTFRIP YYV GPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGG HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLY YLQN GRDM Y VD QELDINRLS D YD VDHI VPQS FLKDDS IDNKVLTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTK AERGGLS ELDKAGFIKRQLVE TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HH AHD AYFN AV V GTAFIKKYPKFES EF V Y GD YKV YD VRKMIAKS EQEIGKATAKYF FY S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDFAT VRKVLS MPQ VNIV KKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKG KSKKFKSVKEFFGITIMERSSFEKNPIGFFEAKGYKEVKKDFIIKFPKYSFFEFENGRK RMFAS AG VLHKGNELALPS KY VNFFYFAS H YEKFKGS PEDNEQKQFF VEQHKH YFD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGVPAAFKYF DTTIDKKRYT S TKE VFD ATFIHQS IT GFYETRIDFS QFGGD (SEQ ID NO: 134).

[0151] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-NRCH. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-NRCH. The SpCas9-NRCH has an amino acid sequence as presented in SEQ ID NO: 135 (underligned residues are mutated relative to SpCas9)

MDKKY S IGLDIGTNS VGWAVITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLFDS GET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIF GNIVDE VAYHEKYPTIYHLRKKLVDS TD KADLRLIYLALAHMIKFRGHFLIEGDLN PDNS D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLI AQLPGEK KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYK EIFFDQS KN G YAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD N GIIPHQIHLGELH AILRRQGDFYPFLKDNREKIEKILTFRIP YYV GPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGG HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLY YLQN GRDM Y VD QELDINRLS D YD VDHI VPQS FLKDDS IDNKVLTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTK AERGGLS ELDKAGFIKRQLVE TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HH AHD AYLN AV V GTALIKKYPKLES EF V Y GD YKV YD VRKMIAKS EQEIGKATAKYF FY S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDFAT VRKVLS MPQ VNIV KKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKY GGFNSPTVAY S VLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTINRKQYNTTKE VLD ATLIRQS ITGLYETRIDLS QLGGD (SEQ ID NO: 135)

[0152] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-NRTH. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-NRTH. The SpCas9-NRTH has an amino acid sequence as presented in SEQ ID NO: 136 (underligned residues are mutated relative to SpCas9)

MDKKY S IGLDIGTNS VGWAVITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLFDS GET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIF GNIVDE VAYHEKYPTIYHLRKKLVDS TD KADLRLIYLALAHMIKFRGHFLIEGDLN PDNS D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLI AQLPGEK KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYK EIFFDQS KN G YAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD N GIIPHQIHLGELH AILRRQGDFYPFLKDNREKIEKILTFRIP YYV GPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGG HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLY YLQN GRDM Y VD QELDINRLS D YD VDHI VPQS FLKDDS IDNKVLTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTK AERGGLS ELDKAGFIKRQLVE TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HH AHD AYLN AV V GTALIKKYPKLES EF V Y GD YKV YD VRKMIAKS EQEIGKATAKYF FY S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDFAT VRKVLS MPQ VNIV KKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKY GGFNSPTVAY S VLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASASVLHKGNELALPSKYVNFLYLASHYEKLKGSSEDNKQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGASAAFKYF DTTIGRKLYT S TKE VLD ATLIHQS IT GLYETRIDLS QLGGD (SEQ ID NO: 136)

[0153] In other embodiments, the napDNAbp of any of the disclosed base editors comprises a Cas9 derived from a Streptococcus macacae, e.g. Streptococcus macacae NCTC 11558, or SmacCas9, or a variant thereof. In some embodiments, the napDNAbp comprises a hybrid variant of SmacCas9 that incorporates an SpCas9 domain with the SmacCas9 domain and is known as Spy-macCas9, or a variant thereof. In some embodiments, the napDNAbp comprises a hybrid variant of SmacCas9 that incorporates an increased nucleolytic variant of an SpCas9 (iSpy Cas9) domain and is known as iSpy-macCas9. Relative to Spymac-Cas9, iSpyMac-Cas9 contains two mutations, R221K and N394K, that were identified by deep mutational scans of Spy Cas9 that raise modification rates of the protein on most targets. See Jakimo el al, bioRxiv, A Cas9 with Complete PAM Recognition for Adenine Dinucleotides (Sep 2018), herein incorporated by reference. Jakimo et al. showed that the hybrids Spy- macCas9 and iSpy-macCas9 recognize a short 5'-NAA-3' PAM and recognized all evaluated adenine dinucleotide PAM sequences and posseseds robust editing efficiency in human cells. Liu et al. engineered base editors containing Spy-mac Cas9, and demonstrated that cytidine and base editors containing Spymac domains can induce efficient C-to-T and A-to-G conversions in vivo. In addition, Liu et al. suggested that the PAM scope of Spy-mac Cas9 may be 5 '-T AAA-3', rather than 5'-NAA-3' as reported by Jakimo et al. See Liu et al. Cell Discovery (2019) 5:58, herein incorporated by reference.

[0154] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to iSpyMac-Cas9. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises iSpyMac-Cas9. The iSpyMac-Cas9 has an amino acid sequence as presented in SEQ ID NO: 137 (R221K and N394K mutations are underlined):

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA

EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI

FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNP

DNS D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RKLENLIAQLPGEKK

NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFL

AAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNG

SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR

KS EETITPWNFEE V VD KG AS AQS FIERMTNFDKNLPNEKVLPKHS LLYE YFT V YNELT

KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH

LFDDKVMKQLKRRRYTGW GRLSRKLIN GIRDKQS GKTILDFLKS DGFANRNFMQLIH

DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK PENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYL

YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN

VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR

QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH

AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS

NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT

EIQT V GQN GGLFDDNPKS PLE VTPS KLVPLKKELNPKKY GG Y QKPTTAYP VLLITDTK

QLIPIS VMNKKQFEQNPVKFLRDRGY QQVGKNDFIKLPKYTLVDIGDGIKRLWAS S KE

IHKGN QLV VS KKS QILLYH AHHLDS DLS ND YLQNHN QQFD VLFNEIIS F S KKCKLGKE

HIQKIENVYSNKKNSASffiELAESFIKLLGFTQLGATSPFNFLGVKLNQKQYKGKKDYI

LPCTEGTLIRQS ITGLYETRVDLS KIGED (SEQ ID NO: 137)

[0155] In other embodiments, the napDNAbp of any of the disclosed base editors is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and have been described, for example, in Makarova K., el al.,“Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements”, Biol Direct. 2009 Aug 25;4:29. doi: 10.1186/1745-6150-4-29, the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp is a Marinitoga piezophila Argunaute (MpAgo) protein. The CRISPR-associated Marinitoga piezophila Argunaute (MpAgo) protein cleaves single- stranded target sequences using 5'-phosphorylated guides. The 5' guides are used by all known Argonautes. The crystal structure of an MpAgo-RNA complex shows a guide strand binding site comprising residues that block 5' phosphate interactions. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for a 5'-hydroxylated guide. See, e.g., Kaya el al.,“A bacterial Argonaute with noncanonical guide RNA specificity”,

Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4057-62, the entire contents of which are hereby incorporated by reference). It should be appreciated that other argonaute proteins may be used, and are within the scope of this disclosure.

[0156] In some embodiments, the napDNAbp is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpfl, C2cl, C2c2, and C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpfl are Class 2 effectors. In addition to Cas9 and Cpfl, three distinct Class 2 CRISPR-Cas systems (C2cl, C2c2, and C2c3) have been described by Shmakov el al.,“Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, C2cl and C2c3, contain RuvC-like endonuclease domains related to Cpfl. A third system, C2c2 contains an effector with two predicated HEPN RNase domains.

Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2cl. C2cl depends on both CRISPR RNA and tracrRNA for DNA cleavage.

Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single- stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpfl. See, e.g., East-Seletsky, et al.,“Two distinct RNase activities of CRISPR- C2c2 enable guide-RNA processing and RNA detection”, Nature, 2016 Oct

13;538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2 is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al.,“C2c2 is a single-component programmable RNA-guided RNA- targeting CRISPR effector”, Science, 2016 Aug 5; 353(6299), the entire contents of which are hereby incorporated by reference.

[0157] The crystal structure of Alicyclobaccillus acidoterrastris C2cl (AacC2cl) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al.,“C2cl-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan 19;65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2cl bound to target DNAs as ternary complexes. See e.g., Yang et al.,“P AM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec 15; 167(7): 1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2cl, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2cl -mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2cl ternary complexes and previously identified Cas9 and Cpfl counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.

[0158] In some embodiments, the napDNAbp may be a C2cl, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2cl protein. In some embodiments, the napDNAbp is a C2c2 protein. In some embodiments, the napDNAbp is a C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2cl, C2c2, or C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring C2cl, C2c2, or C2c3 protein.

[0159] Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (SpCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing base editors provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region ( e.g ., a“editing window” or a“target window”), which is approximately 15 bases upstream of the PAM. See Komor, A.C., et al,

“Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the base editors provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al.,“Engineered CRISPR-Cas9 nucleases with altered PAM

specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al.,“Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

[0160] For example, a napDNAbp domain with altered PAM specificity, such as a domain with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Francisella novicida Cpfl (SEQ ID NO: 138) (D917, E1006, and D1255), which has the following amino acid sequence:

[0161] An additional napDNAbp domain with altered PAM specificity, such as a domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Geobacillus thermodenitrificans Cas9 (SEQ ID NO: 139), which has the following amino acid sequence:

[0162] In some embodiments, the nucleic acid programmable DNA binding protein

(napDNAbp) is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5' phosphorylated ssDNA of ~24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM).

Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al, Nat Biotechnol., 34(7): 768-73 (2016), PubMed PMID: 27136078; Swarts et al., Nature, 507(7491): 258-61 (2014); and Swarts et al., Nucleic Acids Res. 43(10) (2015): 5120-9, each of which is incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided in SEQ ID NO: 140.

[0163] The disclosed base editors may comprise a napDNAbp domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 140), which has the following amino acid sequence:

H. Cas9 circular permutants

[0164] In various embodiments, the base editors disclosed herein may comprise a circular permutant of Cas9.

[0165] The term“circularly permuted Cas9” or“circular permutant” of Cas9 or“CP-Cas9”) refers to any Cas9 protein, or variant thereof, that occurs or has been modify to engineered as a circular permutant variant, which means the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein) have been topically rearranged. Such circularly permuted Cas9 proteins, or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes et al.,“Protein Engineering of Cas9 for enhanced function,” Methods Enzymol, 2014, 546: 491-511 and Oakes et al.,“CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification,” Cell , January 10, 2019, 176: 254-267, and Huang, T.P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626-631 (2019). each of are incorporated herein by reference. Reference is also made to International Application No. PCT/US2019/47996, filed August 23, 2019, herein incorporated by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA). [0166] Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.

[0167] In various embodiments, the circular permutants of Cas9 may have the following structure:

N-terminus-[original C-terminus] - [optional linker] - [original N-terminus]-C-terminus.

[0168] As an example, the present disclosure contemplates the following circular permutants of canonical S. pyogenes Cas9 (1368 amino acids of UniProtKB - Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 9)):

N -terminu s- [ 1268 - 1368 ] - [optional linker] -[1-1267] -C- terminu s ;

N -terminu s- [ 1168 - 1368 ] - [optional linker] -[1-1167] -C- terminu s ;

N -terminu s- [ 1068 - 1368 ] - [optional linker] -[1-1067] -C- terminu s ;

N-terminus- [968- 1368] - [optional linker] - [ 1 -967] -C-terminus;

N -terminu s- [868-1368] - [optional linker] - [l-867]-C -terminu s ;

N -terminu s- [768-1368] - [optional linker] - [ 1 -767 ] -C -terminu s ;

N-terminus- [668- 1368] - [optional linker] - [ 1 -667] -C-terminus;

N -terminu s- [568-1368] - [optional linker] - [l-567]-C -terminu s ;

N -terminu s- [468-1368] - [optional linker] - [l-467]-C -terminu s ;

N -terminu s- [368-1368] - [optional linker] - [l-367]-C -terminu s ;

N -terminu s- [268-1368] - [optional linker] - [l-267]-C -terminu s ;

N -terminu s- [168-1368] - [optional linker] - [1-167]-C -terminu s ;

N-terminus-[68-1368]-[optional linker]-[l-67]-C-terminus; or

N-terminus-[10-1368]-[optional linker]-[l-9]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).

[0169] In particular embodiments, the circular permuant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB - Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 9):

N -terminu s- [102-1368] - [optional linker] - [1-101]-C -terminu s ;

N-terminus-[1028-1368]-[optional linker]-[l-1027]-C-terminus;

N -terminus- [1041-1368] - [optional linker] -[1-1043] -C-terminus ;

N-terminus-[1249-1368]-[optional linker]-[l-1248]-C-terminus; or

N-terminus-[1300-1368]-[optional linker]-[l-1299]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc). [0170] In still other embodiments, the circular permutant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB - Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 9):

N -terminu s- [103-1368] - [optional linker] - [ 1 - 102] -C -terminu s ;

N-terminus-[1029-1368]-[optional linker]-[l-1028]-C-terminus;

N -terminu s- [ 1042- 1368 ] - [optional linker] -[1-1041] -C- terminu s ;

N-terminus-[1250-1368]-[optional linker]-[l-1249]-C-terminus; or

N-terminus-[1301-1368]-[optional linker]-[l-1300]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc.).

[0171] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, The C-terminal fragment may correspond to the C-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1300-1368), or the C-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,

45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9. The N-terminal portion may correspond to the N-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1-1300), or the N-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9 (e.g., of SEQ ID NO: 9).

[0172] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 of SEQ ID NO: 9). In some

embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,

2%, or 1% of the amino acids of a Cas9 (e.g., the Cas9 of SEQ ID NO: 9). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., the Cas9 of SEQ ID NO:

9). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140,

130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 9). In some embodiments, the C-terminal portion that is rearranged to the N- terminus, includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 9).

[0173] In other embodiments, circular permutant Cas9 variants may be defined as a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 9: (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N-terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to preceed the original N- terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue. The CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain. For example, the CP site may be located (relative the S. pyogenes Cas9 of SEQ ID NO: 9) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to the N-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 would become the new N- terminal amino acid. Nomenclature of these CP-Cas9 proteins may be referred to as Cas9- CP¹⁸¹, Cas9-CP¹⁹⁹, Cas9-CP²³⁰, Cas9-CP²⁷⁰, Cas9-CP³¹⁰, Cas9-CP¹⁰¹⁰, Cas9-CP¹⁰¹⁶, Cas9- CP¹⁰²³, Cas9-CP¹⁰²⁹, Cas9-CP¹⁰⁴¹, Cas9-CP¹²⁴⁷, Cas9-CP¹²⁴⁹, and Cas9-CP¹²⁸², respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 9, but may be implemented to make CP variants in any Cas9 sequence, either at CP sites that correspond to these positions, or at other CP sites entireley. This description is not meant to limit the specific CP sites in any way. Virtually any CP site may be used to form a CP-Cas9 variant.

[0174] Exemplary CP-Cas9 amino acid sequences, based on the Cas9 of SEQ ID NO: 9, are provided below in which linker sequences are indicated by underlining and optional methionine (M) residues are indicated in bold. It should be appreciated that the disclosure provides CP-Cas9 sequences that do not include a linker sequence or that include different linker sequences. It should be appreciated that CP-Cas9 sequences may be based on Cas9 sequences other than that of SEQ ID NO: 9 and any examples provided herein are not meant to be limiting. Exemplary CP-Cas9 sequences are as follows:

[0175] The Cas9 circular permutants that may be useful in the base editor constructs described herein. Exemplary C-terminal fragments of Cas9, based on the Cas9 of SEQ ID NO: 9, which may be rearranged to an N-terminus of Cas9, are provided below. It should be appreciated that such C-terminal fragments of Cas9 are exemplary and are not meant to be limiting. These exemplary CP-Cas9 fragments have the following sequences:

I. Cas9 variants with modified PAM specificities

[0176] The base editors of the present disclosure may also comprise Cas9 variants with modified PAM specificities. Some aspects of this disclosure provide Cas9 proteins that exhibit activity on a target sequence that does not comprise the canonical PAM (5'-NGG-3', where N is A, C, G, or T) at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'-NGG-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NNG- 3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'-NNA-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'-NNC-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NNT-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NGT-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NGA-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NGC-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'- NAA-3' PAM sequence at its 3 -end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NAC-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NAT-3' PAM sequence at its 3 -end. In still other embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NAG-3' PAM sequence at its 3 -end.

[0177] In some embodiments, the disclosed base editors comprise a napDNAbp domain comprising a SpCas9-NG, which has a PAM that corresponds to NGN. In some

embodiments, the disclosed base editors comprise a napDNAbp domain comprising a SpCas9-KKH, which has a PAM that corresponds to NNNRRT (SEQ ID NO: 153).

[0178] It should be appreciated that any of the amino acid mutations described herein, (e.g., A262T) from a first amino acid residue (e.g., A) to a second amino acid residue (e.g., T) may also include mutations from the first amino acid residue to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue. For example, mutation of an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) may be a mutation to a second amino acid with a different hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan). For example, a mutation of an alanine to a threonine (e.g., a A262T mutation) may also be a mutation from an alanine to an amino acid that is similar in size and chemical properties to a threonine, for example, serine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with a different positively charged side chain (e.g., arginine, histidine, or lysine). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a different polar side chain (e.g., serine, threonine, asparagine, or glutamine). Additional similar amino acid pairs include, but are not limited to, the following: phenylalanine and tyrosine; asparagine and glutamine; methionine and cysteine; aspartic acid and glutamic acid; and arginine and lysine. The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a threonine may be an amino acid mutation to a serine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an arginine may be an amino acid mutation to a lysine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a lysine may be an amino acid mutation to an arginine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.

[0179] In some embodiments, the present disclosure may utilize any of the Cas9 variants disclosed in the SEQUENCES section herein.

[0180] In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5 -NAA-3' PAM sequence at its 3 - end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 1.

Table 1: NAA PAM Clones

[0181] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1.

[0182] In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5'-NGG-3') at its 3' end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 9. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3' end that is not directly adjacent to the canonical PAM sequence (5'-NGG-3') that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 9 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5'-NGG-3') that is at least 10- fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000- fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 9 on the same target sequence. In some embodiments, the 3' end of the target sequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5 -NAC-3' PAM sequence at its 3 '-end. In some

embodiments, the combination of mutations are present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 2.

Table 2: NAC PAM Clones

[0183] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2.

[0184] In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5'-NAT-3' PAM sequence at its 3 '-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 3. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 3.

Table 3: NAT PAM Clones

[0185] The above description of various napDNAbps which can be used in connection with the presently disclose base editors is not meant to be limiting in any way. The base editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein— including any naturally occurring variant, mutant, or otherwise engineered version of Cas9— that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 varants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other

embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are“dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats). The base editors described herein may also comprise Cas9 equivalents, including Casl2a/Cpfl and Casl2b proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter/enhance their PAM specifities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequences or a reference Cas9 equivalent (e.g., Casl2a/Cpfl).

[0186] In a particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VRQR, or SpCas9-VRQR. In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-VRQR. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-VRQR. The SpCas9- VRQR comprises the following amino acid sequence (with the V, R, Q, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 151 show, in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRQR):

[0187] In another particular embodiment, the Cas9 variant having expanded PAM

capabilities is SpCas9 (H840A) VRER, having the following amino acid sequence (with the V, R, E, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 152 are shown in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRER):

[0188] In addition, any available methods may be utilized to obtain or construct a variant or mutant Cas9 protein. The term“mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include“loss-of-function” mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. Mutations also embrace“gain-of-function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.

[0189] Mutations can be introduced into a reference Cas9 protein using site-directed mutagenesis. Older methods of site-directed mutagenesis known in the art rely on sub cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3 ' end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation. More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.

[0190] Any of the references noted above which relate to napDNAbp domains are hereby incorporated by reference in their entireties, if not already stated so.

II. Thymine alkyltransferases

[0191] In various embodiments, the transversion base editors provided herein comprise a thymine alkyltransferase (FIG. 1A). The thymine alkyltransferase may be modified from its wild type form. Modified thymine alkyltransferases may be obtained by, e.g., evolving a reference version (e.g., an RNA modification enzyme such as a ribosomal RNA

alkyltransferase) using a continuous evolution process (e.g., PACE) or non-continuous evolution process (e.g., PANCE or discrete plate-based selections) described herein so that the alkyltransferase is effective on a nucleic acid target. See Sharma el al, Indentification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m³U methylations of 25S rRNA in Saccharomyces cerevisiae, Nucleic Acid Res. (2014) 42(5): 3246-3260 and Meyer el al, Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans, Nucleic Acid Res. (2016) 44(9): 4304- 4316, the entire contents of each of which is herein incorporated by reference.

[0192] In some embodiments, the nucleobase modification domain is a thymine

alkyltransferase (e.g., RsmE (E. coli )), or an evolved variant thereof.

[0193] The desired thymine alkylation reaction, i.e. the reaction that produces an N3-methyl- thymine, N3-carboxymethyl thymine, or N3-3-amino-3-carboxypropyl thymine product, may be selected based on the relevant enzyme and S - adc n o s y 1 - m c t h i o nine (SAM) cofactor used in the reaction. To yield an N3 -methyl-thymine product, an unmodified SAM is used with an Escherichia coli RsmE, a Saccharomyces cerevisiae Bmt5 or a Saccharomyces cerevisiae Bmt6, or a variant thereof (FIG. IB). To yield an N3-3-amino-3-carboxypropyl thymine product, an unmodified SAM is used with a Tsr3 aminocarboxypropyl transferase, or variant thereof (FIGS. 2A, 2B). To yield an N3-carboxy methyl thymine, a SAM cofactor modified to include a carboxymethyl domain on the S⁺ center may be used (FIG. IB). A variant of an Escherichia coli RsmE, a Saccharomyces cerevisiae Bmt5 or a Saccharomyces cerevisiae Bmt6 that has been evolved using a continuous evolution process (e.g., PACE) to accept a carboxylated SAM cofactor may be used.

[0194] Some exemplary thymine alkyltransferase domains that may be fused to Cas9 domains according to embodiments of this disclosure are provided below. Exemplary thymine alkyltransferase domains include:

E. coli RsmE

[0195] MRIPRIYHPEPLTS HS HIALCED A ANHIGR VLRMGPGQ ALQLFD GS N Q VFD AE ITS AS KKS VE VKVLEGQIDDRES PLHIHLGQ VMS RGEKMEFTIQKS IELG V S LITPLFS E RCGVKLDSERLNKKLQQWQKIAIAACEQCGRNRVPEIRPAMDLEAWCAEQDEGLKL NLHPR AS N S INTLPLP VER VRLLIGPEGGLS ADEIAMT ARY QFTDILLGPR VLRTETT A LTAITALQVRFGDLG (SEQ ID NO: 51)

S. cerevisiae Bmt5

[0196] M ARKLKGKIGS KGLKG ALLRHKAKVKLVRNIES KQKHELRKKN S S ANNKT V KRNQEFQKLNQGKVMPFEKDETLMLCGEGDFSFARSIVEQNYIESDNLIITSYDNSVN ELKLKYPHTFEEN Y Q YLKDLNIPIFF QID VTKLVKS FKIS KNNT WFKIINRLS DHRW GN KPLQNIVFNFPHNGKGIKDQERNIREHQDLIFNFFQNSLQLFNLINTKIQNDTLRYTQG YDLNEDTPQAKKLT AEGY GNIILS LFDGEPYDS WQIKLLAKKN GLTLSRS S KFQWEN FPG YHHRRTN S EQDTTKP AKERD ARF YIF S K Y V S NS S KHNRKS KKDTDS DS D (SEQ ID NO: 52)

S. cerevisiae Bmt6

[0197] MLLMRRF AFLT S S V YFKYIPIY S Q YH Y S S QFPINMNPKKV AQLP VHNKS TLPP QEIIDLFKITFLEELYPKDQDNEKSPLTEQIQAVKSDLYNRDYNAAFNNDSKRIAYCC RWSPSRATSYASVFAHFPELLKIIRCEIDDKDSNVLCIGGGAGGELVALASIFTLSRDF SSKFASALKIDNEVNKKPRNLNIQLVDIADWSTVVEKLTATIKSKWLYGDSEAESFN VNFTHKDCLQMTEPQDIKIYQGLDLITLLFTTNELFTQKKVESIKFLQRLNENCAPGC HLLILES AGS Y S HITINNKKFPIQFLIDTILV GNRKDKGTTGPW S LV S ENDS IW YRMDP KLD Y S IPLENMRFFYRLY VKN (SEQ ID NO: 53) AlkBHl (human)

[0198] MGKM A A A V GS V ATL ATEPGED AFRKLFRF YRQS RPGT ADLEG VIDFS A AH A ARGKGPG AQKVIKS QLN V S S V S EQN A YRAGLQP V S KW Q A Y GLKG YPGFIFIPNPFLP GYQWHWVKQCLKLYSQKPNVCNLDKHMSKEETQDLWEQSKEFLRYKEATKRRPR S LLEKLRW VT V G YH YNWDS KKY S ADH YTPFPS DLGFLS EQ V A A AC GFEDFR AE AGI LN Y YRLDS TLGIH VDRS ELDHS KPLLS FS F GQS AIFLLGGLQRDE APT AMFMHS GDIM IMSGFSRLLNHAVPRVLPNPEGEGLPHCLEAPLPAVLPRDSMVEPCSMEDWQVCASY LKTARVNMTVRQVLATDQNFPLEPIEDEKRDISTEGFCHLDDQNSEVKRARINPDS

(SEQ ID NO: 54)

AlkBH2 (human)

[0199] MDRFLVKGAQGGLLRKQEEQEPTGEEPAVLGGDKESTRKRPRREAPGNGGH S AGPS WRHIR AEGLDC S YT VLFGKAE ADEIF QELEKE VE YFT G ALAR V Q VF GKWHS VPRKQATYGDAGLTYTFSGLTLSPKPWIPVLERIRDHVSGVTGQTFNFVLINRYKDG CDHIGEHRDDERELAPGS PIAS VS F G ACRDF VFRHKDS RGKS PS RRV A V VRLPLAHGS LLMMNHPTNTHWYHSLPVRKKVLAPRVNLTFRKILLTKK (SEQ ID NO: 55)

AlkBH3 (human):

[0200] MEEKRRR ARV QG A W A AP VKS Q AIAQP ATT AKS HLHQKPGQT WKNKEHHLS DREFVFKEPQQVVRRAPEPRVIDREGVYEISLSPTGVSRVCLYPGFVDVKEADWILEQ LCQDVPWKQRTGIREDITY QQPRLTAWY GELPYTYSRITMEPNPHWHPVLRTLKNRI EENTGHTFN S LLCNLYRNEKDS VD WHS DDEPS LGRCPIIAS LS F G ATRTFEMRKKPPP EENGDYTYVERVKIPLDHGTLLIMEGATQADWQHRVPKEYHSREPRVNLTFRTVYP DPRGAPW (SEQ ID NO: 56)

AlkBH4 (human)

[0201] MAAAAAETPEVLRECGCKGIRTCLICERQRGSDPPWELPPAKTYRFIYCSDTG W A V GTEES DFEGW AFPFPG VMLIEDF VTREEE AELVRLMDRDPWKLS QS GRRKQD Y GPKVNFRKQKLKTEGFCGLPSFSREVVRRMGLYPGLEGFRPVEQCNLDYCPERGSAI DPHLDD A WLW GERLV S LNLLS PT VLS MCRE APGS LLLC S APS A APE ALVDS VIAPS R S VLCQE VE V AIPLP ARS LLVLT G A ARHQWKH AIHRRHIE ARRVC VTFRELS AEF GPG GRQQELGQELLRIALS FQGRPV (SEQ ID NO: 57)

AlkBH5 (human)

[0202] M AA AS G YTDLREKLKS MTS RDN YKAGS RE A A A A A A AAV A A A A A AAA A AE PYPV S GAKRKY QEDSDPERSD YEEQQLQKEEEARKVKS GIRQMRLFS QDEC AKIE AR IDEVVSRAEKGLYNEHTVDRAPLRNKYFFGEGYTYGAQLQKRGPGQERLYPPGDVD EIPEWVHQLVIQKLVEHRVIPEGFVNSAVINDYQPGGCIVSHVDPIHIFERPIVSVSFFS DSALCFGCKFQFKPIRVSEPVLSLPVRRGSVTVLSGYAADEITHCIRPQDIKERRAVIIL RKTRLD APRLETKS LS S S VLPPS Y AS DRLS GNNRDP ALKPKRS HRKADPD A AHRPRIL EMDKEENRRS VLLPTHRRRGS FS S EN YWRKS YES S EDC S E A AGS P ARKVKMRRH

(SEQ ID NO: 58)

AlkBH6 (human)

[0203] MEEQDARVPALEPFRVEQAPPVIYYVPDFISKEEEEYLLRQVFNAPKPKWTQL S GRKLQNW GGLPHPRGMVPERLPPWLQRY VDKVSNLS LFGGLPANHVLVN Q YLPG EGIMPHEDGPLY YPT V S TIS LGS HT VLDFYEPRRPEDDDPTEQPRPPPRPTT S LLLEPRS LLVLRGP A YTRLLHGI A A ARVD ALD A AS S PPN A A ACPS ARPG ACLVRGTRV S LTIRR VPRVLRAGLLLGK (SEQ ID NO: 59)

AlkBH7 (human)

[0204] M AGT GLLALRTLPGPS W VRGS GPS VLS RLQD A A V VRPGFLS T AEEETLS RELE PELRRRRYE YDHWD A AIHGFRETEKS RW SEAS RAILQRV Q A A AF GPGQTLLS S VH VL DLEARGYIKPHVDSIKFCGATIAGLSLLSPSVMRLVHTQEPGEWLELLLEPGSLYILRG SARYDFSHEILRDEESFFGERRIPRGRRISVICRSLPEGMGPGESGQPPPAC (SEQ ID NO: 60)

AlkBH8 (human)

[0205] MDS NHQS N YKLS KTEKKFLRKQIKAKHTLLRHEGIET V S Y ATQS LV V AN GGL GN GVSRN QLLPVLEKCGLVD ALLMPPNKPYSFARYRTTEES KRA YVTLN GKEVVDD LGQKITLYLNF VEKV QWKELRPQ ALPPGLM V VEEIIS S EEEKMLLES VD WTEDTDN Q NSQKSLKHRRVKHFGYEFHYENNNVDKDKPLSGGLPDICESFLEKWLRKGYIKHKP DQMTINQYEPGQGIPAHIDTHSAFEDEIVSLSLGSEIVMDFKHPDGIAVPVMLPRRSLL VMT GES R YLWTHGITCRKFDT V Q AS ES LKS GUTS D V GDLTLS KRGLRT S FTFRKVRQ TPCNCSYPLVCDSQRKETPPSFPESDKEASRLEQEYVHQVYEEIAGHFSSTRHTPWPH IVEFLKALPSGSIVADIGCGNGKYLGINKELYMIGCDRSQNLVDICRERQFQAFVCDA LA VP VRS GS CD AC IS I A VIHHFAT AERRV A ALQEIVRLLRPGGKALIY VW AMEQE YN KQKSKYLRGNRNSQGKKEEMNSDTSVQRSLVEQMRDMGSRDSASSVPRINDSQEG GCN S RQ V S NS KLP VH VNRTS F Y S QD VLVPWHLKGNPDKGKP VEPF GPIGS QDPS P VF HRYYHVFREGELEGACRTVSDVRILQSYYDQGNWCVILQKA (SEQ ID NO: 61)

Tsr3 (human)

[0206] MGRRRAARGPGAEGGRPRHLPTRSLEAFAEEVGAALQASVEPGAADGEGGP GPAALPCTLAMWELGHCDPRRCTGRKLARLGLVRCLRLGHRFGGLVLSPVGKQYA S P ADRQLV AQS G V A VIDC S W ARLDETPF GKMRGS HLRLLP YLV A ANP VN Y GRP YRL SCVEAFAATFCIVGFPDLAVILLRKFKWGKGFLDLNRQLLDKYAACGSPEEVLQAEQ EFLAN AKES PQEEEIDPFD VDS GREF GNPNRP V AS TRLPS DTDDS D AS EDPGPG AERG GAS S SCCEEEQTQGRGAEARAPAEVWKGIKKRQRD (SEQ ID NO: 62)

S. cerevisiae T sr3

[0207] MGKGKNKMHEPKN GRPQRG AN GHS S RQNHRRMEMKYDN S EKMKFP VKLA M WDFDHCDPKRC S GKKLERLGLIKS LR V GQKF QGIV V S PN GKG V V CPDDLEIVEQH GAS V VEC S W ARLEE VPFNKIGGKHERLLP YL V A AN Q VN Y GRPWRLNC VE ALA ACF AIVGRMDWASELLSHFSWGMGFLELNKELLEIYQQCTDCDSVKRAEEEWLQKLEKE TQERKSRAKEEDIWMMGNINRRGNGSQSDTSESEENSEQSDLEGNNQCIEYDSLGNA IRIDNMKS RE AQS EES EDEES GS KEN GEPLS YDPLGNLIR (SEQ ID NO: 63)

V. distributa Tsr3

[0208] MIPR VFIYRLPQDDPRKNT AIKLVRF GF AQL VDS IKALPS GS IILDPT VKTPLTP S DRVIAES RGLS LIDC S WKR A VD VHTKFIRGKFIRRRLPLLIA ANPTH Y GKP YILS TIE A VAAALYIMGFKDEAMEVLRLYKWGPNFIIINQKYLERYAAGDLSPERELLGVDDVD N GLEQLMRVLTN GD (SEQ ID NO: 64)

S. solfataricus Tsr3

[0209] MKVYIIDYHKDDPKRCTGKKLVKLKIAEFTRVGKGVVLDPFAQITLSNKDKDI VRRIGITIVDTSWNNTSQSEFKNIRGEHRRIPILFAGNPIHYGIAYKLSSIEALIATLYIV DEVEEAIKLSNVVKWGHTFIELNKELLEAYKNKTEEDIKKIEREIIEKILEK (SEQ ID NO: 65)

Mouse DNMT1

[0210] MPART AP AR VP ALAS P AGS LPDH VRRRLKDLERDGLTEKEC VREKLNLLHEF

LQTEIKSQLCDLETKLHKEELSEEGYLAKVKSLLNKDLSLENGTHTLTQKANGCPAN

GS RPT WRAEM ADS NRS PRS RPKPRGPRRS KS DS DTLS VET S PS S V ATRRTTRQTTIT A

HFTKGPTKRKPKEESEEGNSAESAAEERDQDKKRRVVDTESGAAAAVEKLEEVTAG

TQLGPEEPCEQEDDNRSLRRHTRELSLRRKSKEDPDREARPETHLDEDEDGKKDKRS

SRPRSQPRDPAAKRRPKEAEPEQVAPETPEDRDEDEREEKRRKTTRKKLESHTVPVQ

SRSERKAAQSKSVIPKINSPKCPECGQHLDDPNLKYQQHPEDAVDEPQMLTSEKLSIY

DSTSTWFDTYEDSPMHRFTSFSVYCSRGHLCPVDTGLIEKNVELYFSGCAKAIHDENP

SMEGGINGKNLGPINQWWLSGFDGGEKVLIGFSTAFAEYILMEPSKEYEPIFGLMQEK

IYIS KIV VEFLQNNPD A V YEDLINKIETT VPPS TIN VNRFTEDS LLRH AQF V V S Q VES YD

EAKDDDETPIFLSPCMRALIHLAGVSLGQRRATRRVMGATKEKDKAPTKATTTKLV

YQIFDTFFSEQIEKYDKEDKENAMKRRRCGVCEVCQQPECGKCKACKDMVKFGGT

GRSKQACLKRRCPNLAVKEADDDEEADDDVSEMPSPKKLHQGKKKKQNKDRISWL

GQPMKIEENRT Y Y QK V S IDEEMLE V GDC V S VIPDDS S KPLYLARVT ALWED KN GQM MFH AHWFC AGTDT VLG AT S DPLELFLV GECENMQLS YIHS KVKVI YKAPS ENW AME GGTDPETTLPGAEDGKTYFFQLWYNQEYARFESPPKTQPTEDNKHKFCLSCIRLAEL RQKEMPKVLEQIEEVDGRVYCSSITKNGVVYRLGDSVYLPPEAFTFNIKVASPVKRP KKDPVNETLYPEHYRKYSDYIKGSNLDAPEPYRIGRIKEIHCGKKKGKVNEADIKLRL YKFYRPENTHRS YNGS YHTDINMLYW S DEE A V VNF S D V QGRCT VE Y GEDLLES IQD Y S QGGPDRF YFLE A YN S KTKNFEDPPNH ARS PGNKGKGKGKGKGKGKHQ V S EPKEP EAAIKLPKLRTLDVFSGCGGLSEGFHQAGISETLWAIEMWDPAAQAFRLNNPGTTVF TEDCN VLLKLVM AGE VTN S LGQRLPQKGD VEMLC GGPPCQGF S GMNRFN S RT Y S K FKN S LVVSFLS YCD YYRPRFFLLENVRNFV S YRRSMVLKLTLRCLVRMGY QCTFGV LQ AGQ Y G V AQTRRR AULA A APGEKLPLFPEPLH VF APRAC QLS V V VDDKKFV S NIT RLS S GPFRTIT VRDTMS DLPEIQN GAS N S EIP YN GEPLS WF QRQLRGS H Y QPILRDHIC KDMSPLVAARMRHIPLFPGSDWRDLPNIQVRLGDGVIAHKLQYTFHDVKNGYSSTG ALRGVCSCAEGKACDPESRQFSTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTV TNPEPMGKQGRVLHPEQHRVVSVRECARSQGFPDSYRFFGNILDRHRQVGNAVPPPL AKAIGLEIKLCLLS S ARES AS A A VKAKEE A ATKD (SEQ ID NO: 70)

Human DNMT1

[0211] MPARTAPARVPTLAVPAISLPDDVRRRLKDLERDSLTEKECVKEKLNLLHEFL

QTEIKN QLCDLETKLRKEELSEEGYLAKVKS LLNKDLS LEN GAHA YNREVN GRLEN

GN Q ARS E ARR V GM AD AN S PPKPLS KPRTPRRS KS DGE AKPEPS PS PRITRKS TRQTTIT

S HFAKGP AKRKPQEES ERAKS DES IKEEDKD QDEKRRR VT S RER V ARPLP AEEPER A

KS GTRTEKEEERDEKEEKRLRS QTKEPTPKQKLKEEPDREARAGV QADEDEDGDEK

DEKKHRSQPKDLAAKRRPEEKEPEKVNPQISDEKDEDEKEEKRRKTTPKEPTEKKMA

RAKT VMN S KTHPPKCIQC GQ YLDDPDLK Y GQHPPD A VDEPQMLTNEKLS IFD ANES

GFESYEALPQHKLTCFSVYCKHGHLCPIDTGLIEKNIELFFSGSAKPIYDDDPSLEGGV

NGKNLGPINEWWITGFDGGEKALIGFSTSFAEYILMDPSPEYAPIFGLMQEKIYISKIV

VEFLQSNSDSTYEDLINKIETTVPPSGLNLNRFTEDSLLRHAQFVVEQVESYDEAGDS

DEQPIFLTPCMRDLIKLAGVTLGQRRAQARRQTIRHSTREKDRGPTKATTTKLVYQIF

DTFFAEQIEKDDREDKENAFKRRRCGVCEVCQQPECGKCKACKDMVKFGGSGRSK

QACQERRCPNMAMKEADDDEEVDDNIPEMPSPKKMHQGKKKKQNKNRISWVGEA

VKTDGKKSYYKKVCIDAETLEVGDCVSVIPDDSSKPLYLARVTALWEDSSNGQMFH

AHWFCAGTDTVLGATSDPLELFLVDECEDMQLSYIHSKVKVIYKAPSENWAMEGG

MDPESLLEGDDGKTYFYQLWYDQDYARFESPPKTQPTEDNKFKFCVSCARLAEMRQ

KEIPRVLEQLEDLDSRVLYYSATKNGILYRVGDGVYLPPEAFTFNIKLSSPVKRPRKE

PVDEDLYPEHYRKYSDYIKGSNLDAPEPYRIGRIKEIFCPKKSNGRPNETDIKIRVNKF YRPENTHKSTPAS YHADINLLYWSDEEAVVDFKAVQGRCTVEY GEDLPECV QVYSM GGPNRF YFLE A YN AKS KS FEDPPNH ARS PGNKGKGKGKGKGKPKS Q AC EPS EPEIEI KLPKLRTLD VF S GCGGLS EGFHQ AGIS DTLW AIEMWDP A AQ AFRLNNPGS T VFTEDC NILLKLVM AGETTN S RGQRLPQKGD VEMLC GGPPCQGF S GMNRFN S RT Y S KFKN S L VVSFLSYCDYYRPRFFLLENVRNFVSFKRSMVLKLTLRCLVRMGYQCTFGVLQAGQ YGVAQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKKFVSNITRLSSGP FRTITVRDTMSDLPEVRNGASALEISYNGEPQSWFQRQLRGAQYQPILRDHICKDMS ALV A ARMRHIPLAPGS D WRDLPNIE VRLS DGTM ARKLRYTHHDRKN GRS S S G ALRG VCSCVEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNP EPMGKQGRVLHPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPPLAK AIGLEIKLCMLAKARESASAKIKEEEAAKD (SEQ ID NO: 71)

Human DNMT3a

[0212] MPAMPS S GPGDT S S S AAEREEDRKDGEEQEEPRGKEERQEPSTT ARKV GRPG

RKRKHPP VES GDTPKDP A VIS KS PS M AQDS GAS ELLPN GDLEKRS EPQPEEGS P AGGQ

KGGAPAEGEGAAETLPEASRAVENGCCTPKEGRGAPAEAGKEQKETNIESMKMEGS

RGRLRGGLGWESSLRQRPMPRLTFQAGDPYYISKRKRDEWLARWKREAEKKAKVI

AGMNAVEENQGPGESQKVEEASPPAVQQPTDPASPTVATTPEPVGSDAGDKNATKA

GDDEPEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWF

GDGKFSVVCVEKLMPLSSFCSAFHQATYNKQPMYRKAIYEVLQVASSRAGKLFPVC

HDSDESDTAKAVEVQNKPMIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEP

EAAAYAPPPPAKKPRKSTAEKPKVKEIIDERTRERLVYEVRQKCRNIEDICISCGSLNV

TLEHPLFVGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRC

FCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANN

HDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSIT

V GMVRHQGKIM YV GD VRS VTQKHIQEW GPFDLVIGGSPCNDLS IVNPARKGLYEGT

GRLFFEFYRLLHD ARPKEGDDRPFFWLFEN V V AMG V S DKRDIS RFLES NP VMID AKE

VSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIK

QGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVP

VIRHLFAPLKEYFACV (SEQ ID NO: 72)

Human DNMT3b

[0213] MKGDTRHLNGEEDAGGREDSILVNGACSDQSSDSPPILEAIRTPEIRGRRSSSR LSKREVSSLLSYTQDLTGDGDGEDGDGSDTPVMPKLFRETRTRSESPAVRTRNNNSV S S RERHRPS PRS TRGRQGRNH VDES P VEFP ATRS LRRRAT AS AGTPWPSPPS S YLTIDL TDDTEDTHGTPQS S STPY ARLAQDS QQGGMESPQVE ADS GDGDS SEY QDGKEFGIG DLVWGKIKGFSWWPAMVVSWKATSKRQAMSGMRWVQWFGDGKFSEVSADKLVA LGLF S QHFNLATFNKLV S YRKAM YH ALEK ARVR AGKTFPS S PGDS LEDQLKPMLEW AHGGFKPTGIEGLKPNNT QP V VNKS KVRR AGS RKLES RKYENKTRRRT ADDS AT S D Y CP APKRLKTNC YNN GKDRGDEDQS REQM AS D V ANNKS S LEDGCLS C GRKNPV S F HPLFEGGLCQTCRDRFLELFYMYDDDGYQSYCTVCCEGRELLLCSNTSCCRCFCVEC LE VLV GT GT A AE AKLQEPW S C YMCLPQRCHG VLRRRKD WN VRLQ AFFT S DT GLE Y EAPKLYPAIPAARRRPIRVLSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVK HEGNIKYVNDVRNITKKNIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEF YHLLNYSRPKEGDDRPFFWMFENVVAMKVGDKRDISRFLECNPVMIDAIKVSAAHR ARYFW GNLPGMNRPVIAS KNDKLELQDCLEYNRIAKLKKV QTITTKSNS IKQGKN QL FPVVMNGKEDVLWCTELERIFGFPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFA PLKDYFACE (SEQ ID NO: 73)

Hhal methyltransferase from Haemophilus parahaemolyticus

[0214] MIEIKDKQLTGLRFIDLFAGLGGFRLALESCGAECVYSNEWDKYAQEVYEMN FGEKPEGDITQVNEKTIPDHDILCAGFPCQAFSISGKQKGFEDSRGTLFFDIARIVREK KPKV VFMEN VKNF AS HDN GNTLE V VKNTMNELD Y S FH AKVLN ALD Y GIPQKRERIY MICFRNDLNIQNFQFPKPFELNTFVKDLLLPDSEVEHLVIDRKDLVMTNQEIEQTTPK TVRLGIVGKGGQGERIYSTRGIAITLSAYGGGIFAKTGGYLVNGKTRKLHPRECARV MGYPDS YKVHPS TS Q A YKQF GN S V VIN VLQ YIA YNIGS S LNFKP Y (SEQ ID NO: 74) CpG Methyltransferase (M.SssI) from Spiroplasma monobiae

[0215] MSKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMY QAIHNNFHTKLEYKS VSREEMID YLENKTLS WN S KNPVSNGYWKRKKDDELKIIYN AIKLS EKEGNIFDIRDLYKRTLKNIDLLT YSFPCQDLS QQGIQKGMKRGS GTRS GLLW EIERALDSTEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLNA ADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTE FKKTKS NINKAS LIG Y S KFN S EG Y V YDPEFT GPTLT AS GAN S RIKIKDGS NIRKMN S DE TFT .YIGFDSQDGKR VNEIEFI TFNOK IFVGGNS fSVFVI EATTDKTGG (SEQ ID NO: 75)

[0216] In various embodiments, the disclosed fusion proteins comprise a thymine

alkyltransferase domain that does not comprise an E. coli DNA adenine methyltransferase (Dam), or a variant thereof. In some embodiments, the disclosed fusion proteins comprise a thymine alkyltransferase domain that does not comprise a DNA (cytosine-5)- methyltransferase 1 (or DNMT1). In some embodiments, the disclosed fusion proteins comprise a thymine alkyltransferase domain that does not comprise a variant of a DNMT1. In some embodiments, the disclosed fusion proteins do not comprise an E. coli Dam or an DNMT1, or a variant thereof.

III. Additional base editor elements

[0217] In various embodiments, the base editors and constructs encoding the base editors disclosed herein further comprise one or more additional base editor elements, e.g., a nuclear localization signal(s), an inhibitor of base excision repair, and/or a heterologous protein domain.

[0218] In various embodiments, the base editors and constructs encoding the base editors disclosed herein further comprise one or more, preferably, at least two nuclear localization signals. In certain embodiments, the base editors comprise at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLSs or they can be different NLSs. In addition, the NLSs may be expressed as part of a fusion protein with the remaining portions of the base editors. In some embodiments, one or more of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments, the disclosed fusion proteins comprise two bipartite NLSs. In some embodiments, the disclosed fusion proteins comprise more than two bipartite NLSs.

[0219] The location of the NLS fusion can be at the N-terminus, the C-terminus, or within a sequence of a base editor (e.g., inserted between the encoded napDNAbp component (e.g., Cas9) and a DNA nucleobase modification domain (e.g., a thymine alkyltransferase)).

[0220] The NLSs may be any known NLS sequence in the art. The NLSs may also be any future-discovered NLSs for nuclear localization. The NLSs also may be any naturally- occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations).

[0221] The term“nuclear localization sequence” or“NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., International PCT Application No. PCT/EP2000/011690, filed November 23, 2000, published as

WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference. In some embodiments, an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 32), MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 33), KRT ADGS EFES PKKKRKV (SEQ ID NO: 76), or KRT ADGS EFEPKKKRKV (SEQ ID NO: 7). In other embodiments, NLS comprises the amino acid sequences NLS KRP A AIKKAGQ AKKKK (SEQ ID NO: 16), PAAKRVKLD (SEQ ID NO: 17), RQRRNELKRS F (SEQ ID NO: 18),

N QS SNFGPMKGGNFGGRS S GPY GGGGQYFAKPRN QGGY (SEQ ID NO: 19).

[0222] In one aspect of the disclosure, a base editor may be modified with one or more nuclear localization signals (NFS), preferably at least two NFSs. In certain embodiments, the base editors are modified with two or more NFSs. The disclosure contemplates the use of any nuclear localization signal known in the art at the time of the disclosure, or any nuclear localization signal that is identified or otherwise made available in the state of the art after the time of the instant filing. A representative nuclear localization signal is a peptide sequence that directs the protein to the nucleus of the cell in which the sequence is expressed. A nuclear localization signal is predominantly basic, can be positioned almost anywhere in a protein's amino acid sequence, generally comprises a short sequence of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem. 273: 14731-37, incorporated herein by reference) to eight amino acids, and is typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274: 11-16, incorporated herein by reference). Nuclear localization signals often comprise proline residues. A variety of nuclear localization signals have been identified and have been used to effect transport of biological molecules from the cytoplasm to the nucleus of a cell. See, e.g., Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moede et al., (1999) FEBS Lett. 461:229-34, which is incorporated by reference. Translocation is currently thought to involve nuclear pore proteins.

[0223] Most NFSs can be classified in three general groups: (i) a monopartite NFS exemplified by the SV40 large T antigen NFS (PKKKRKV (SEQ ID NO: 32)); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NFS (KRXXXXXXXXXXKKKF (SEQ ID NO: 35)); and (iii) noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NFS, and the yeast Gal4 protein NFS (Dingwall and Faskey 1991).

[0224] Nuclear localization signals appear at various points in the amino acid sequences of proteins. NFS’s have been identified at the N-terminus, the C-terminus and in the central region of proteins. Thus, the disclosure provides base editors that may be modified with one or more NFSs at the C-terminus, the N-terminus, as well as at in internal regaion of the base editor. The residues of a longer sequence that do not function as component NFS residues should be selected so as not to interfere, for example tonically or sterically, with the nuclear localization signal itself. Therefore, although there are no strict limits on the composition of an NLS -comprising sequence, in practice, such a sequence can be functionally limited in length and composition.

[0225] The present disclosure contemplates any suitable means by which to modify a base editor to include one or more NLSs. In one aspect, the base editors may be engineered to express a base editor protein that is translationally fused at its N-terminus or its C-terminus (or both) to one or more NLSs, i.e., to form a base editor-NLS fusion construct. In other embodiments, the base editor-encoding nucleotide sequence may be genetically modified to incorporate a reading frame that encodes one or more NLSs in an internal region of the encoded base editor. In addition, the NLSs may include various amino acid linkers or spacer regions encoded between the base editor and the N-terminally, C-terminally, or internally- attached NLS amino acid sequence, e.g, and in the central region of proteins. Thus, the present disclosure also provides for nucleotide constructs, vectors, and host cells for expressing fusion proteins that comprise a base editor and one or more NLSs.

[0226] The base editors described herein may also comprise nuclear localization signals which are linked to a base editor through one or more linkers, e.g., and polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker element. The linkers within the contemplated scope of the disclosure are not intented to have any limitations and can be any suitable type of molecule (e.g., polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain) and be joined to the base editor by any suitable strategy that effectuates forming a bond (e.g., covalent linkage, hydrogen bonding) between the base editor and the one or more NLSs.

[0227] In certain embodiments, the base editors described herein may comprise an inhibitor of base repair. The term“inhibitor of base repair” or“IBR” refers to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme. In some embodiments, the IBR is an inhibitor of DNA alkylation repair (“iDAR”). In some embodiments, the IBR is an inhibitor of OGG base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some

embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is an iDAR that may be a catalytically inactive glycosylase or catalytically inactive dioxygenase or a small molecule or peptide inhibitor of thymine alkyltransferase, or variants threreof. In some embodiments, the IBR is an iDAR that may be a TDG inhibitor, MBD4 inhibitor or an inhibitor of an AlkBH enzyme. In some embodiments, the IBR is an iDAR that comprises a catalytically inactive TDG or catalytically inactive MBD4. An exemplary catalytically inactive TDG is an N140A mutant of SEQ ID NO: 69 (human TDG).

[0228] Some exemplary glycosylases are provided below. The catalytically inactivated variants of any of these glycosylase domains are iDARs may be fused to the napDNAbp or thymine alkyltransferase domains of the base editors provided in this disclosure.

OGG (human)

[0229] MPAR ALLPRRMGHRTLAS TP ALW AS IPCPRS ELRLDLVLPS GQS FRWREQS PA

HWSGVLADQVWTLTQTEEQLHCTVYRGDKSQASRPTPDELEAVRKYFQLDVTLAQ

LYHHWGSVDSHFQEVAQKFQGVRLLRQDPIECLFSFICSSNNNIARITGMVERLCQAF

GPRLIQLDDVTYHGFPSLQALAGPEVEAHLRKLGLGYRARYVSASARAILEEQGGLA

WLQQLRESSYEEAHKALCILPGVGTKVADCICLMALDKPQAVPVDVHMWHIAQRD

Y S WHPTTS Q AKGPS PQTNKELGNFFRS LW GP Y AGW AQ A VLFS ADLRQS RH AQEPP A

KRRKGS KGPEG (SEQ ID NO: 66)

MPG (human)

[0230] M VTPALQMKKPKQFCRRMGQKKQRPARAGQPHS S SD AAQAPAEQPHS S SD A AQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVR RLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMFMKPGTLYVYIIYGMYF CMNIS S QGDG AC VLLR ALEPLEGLETMRQLRS TLRKGT AS RVLKDRELC S GPS KLCQ ALAINKS FDQRDLAQDE A VWLERGPLEPS EP A V V A A ARV G V GH AGE W ARKPLRF Y VRGS PW V S V VDRV AEQDTQ A (SEQ ID NO: 67)

MBD4 (human)

[0231] MGTTGLESLSLGDRGAAPTVTSSERLVPDPPNDLRKEDVAMELERVGEDEEQ MMIKRS S ECNPLLQEPIAS AQF G AT AGTECRKS VPCGWER V VKQRLF GKT AGRFD V YFIS PQGLKFRS KS S LAN YLHKN GET S LKPEDFDFT VLS KRGIKS RYKDCS M A ALT S H LQN QS NN S NWNLRTRS KCKKD VFMPPS S S S ELQES RGLS NFTS THLLLKEDEG VDD V NFRKVRKPKGKVTILKGIPIKKTKKGCRKS CS GF V QS DS KRES V CNKAD AES EP V AQ KS QLDRT VCIS D AG ACGETLS VT S EENS LVKKKERS LS S GS NFC S EQKT S GIINKFC S A KDSEHNEKYEDTFLESEEIGTKVEVVERKEHLHTDILKRGSEMDNNCSPTRKDFTGE KIF QEDTIPRT QIERRKT S LYF S S KYNKE ALS PPRRKAFKKWTPPRSPFNLV QETLFHD PWKLLIATIFLNRTSGKMAIPVLWKFLEKYPSAEVARTADWRDVSELLKPLGLYDLR AKTIVKFSDEYLTKQWKYPIELHGIGKYGNDSYRIFCVNEWKQVHPEDHKLNKYHD WLWENHEKLS LS (SEQ ID NO: 68) TDG (human)

[0232] ME AEN AGS YS LQQ AQ AF YTFPF QQLM AE APNM A V VNEQQMPEE VP AP AP A QEPV QE APKGRKRKPRTTEPKQPVEPKKPVES KKS GKS AKS KEKQEKITDTFKVKRK VDRFNGVSEAELLTKTLPDILTFNLDIVIIGINPGLMAAYKGHHYPGPGNHFWKCLFM S GLSEV QLNHMDDHTLPGKY GIGFTNM VERTTPGS KDLS S KEFREGGRILV QKLQKY QPRIAVFNGKCIYEIFSKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQFPR AQDKVHYYIKLKDLRDQLKGIERNMD V QE V QYTFDLQLAQED AKKM A VKEEKYDP GYEAAYGGAYGENPCSSEPCGFSSNGLIESVELRGESAFSGIPNGQWMTQSFTDQIPS FSNHCGTQEQEEESHA (SEQ ID NO: 69)

[0233] In some embodiments, the fusion proteins described herein may comprise one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the base editor components). A fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.

Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins.

[0234] Examples of protein domains that may be fused to a fusion protein or component thereof (e.g., the napDNAbp domain, the nucleobase modification domain, or the NLS domain) include, without limitation, epitope tags, and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta- glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A base editor may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a base editor are described in US Patent Publication No. 2011/0059502, published March 10, 2011 and incorporated herein by reference in its entirety. [0235] In an aspect of the disclosure, a reporter gene which includes, but is not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol

acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), may be introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In certain embodiments of the disclosure the gene product is luciferase. In a further embodiment of the disclosure the expression of the gene product is decreased.

[0236] Suitable protein tags provided herein include, but are not limited to, biotin

carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags,

hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S -transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags , biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

IV. Linkers

[0237] In certain embodiments, linkers may be used to link any of the peptides or peptide domains or domains of the disclosure (e.g., domain A covalently linked to domain B which is covalently linked to domain C).

[0238] As defined above, the term“linker,” as used herein, refers to a chemical group or a molecule linking two molecules or domains, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of a napDNAbp nuclease and the catalytic domain of a recombinase. In some embodiments, a linker joins a dCas9 and base editor domain (e.g., a thymine alkyltransferase). Typically, the linker is positioned between, or flanked by, two groups, molecules, or other domains and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical domain. Chemical domains include, but are not limited to, disulfide, hydrazone, thiol and azo domains. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45- 50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some emdobiments, the linker is a single atom, or a single angstrom, in length. Longer or shorter linkers are also contemplated.

[0239] The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or hetero aliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5- pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic domain (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol domain (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain

embodiments, the linker comprises an aryl or heteroaryl domain. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized domains to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0240] In some other embodiments, the linker comprises the amino acid sequence (GGGGS)_n (SEQ ID NO: 43), (G)„ (SEQ ID NO: 44), (EAAAK)„ (SEQ ID NO: 45), (GGS)„ (SEQ ID NO: 46), (SGGS)_n (SEQ ID NO: 47), (XP)_n (SEQ ID NO: 48), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)_n (SEQ ID NO: 34), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 49). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), also known as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 6). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 8).

[0241] In some embodiments, the fusion protein comprises the structure [thymine alkyltransferase]- [optional linker sequence]-[dCas9 or Cas9 nickase]- [optional linker sequence], or [dCas9 or Cas9 nickase] -[optional linker sequence] -[thymine alkyltransferase] - [optional linker sequence] .

[0242] In other embodiments within this same strategy, the fusion protein comprises the structure [thymine alkyltransferase] -[optional linker sequence]-[dCas9 or Cas9 nickase]- [optional linker sequence] -[iDAR]; [thymine alkyltransferase] -[optional linker sequence] - [iDAR]- [optional linker sequence] -[dCas9 or Cas9 nickase]; [iDAR] -[optional linker sequence] -[thymine alkyltransferase] -[optional linker sequence]-[dCas9 or Cas9 nickase]; [iDAR] -[optional linker sequence] -[dCas9 or Cas9 nickase] -[optional linker sequence]- [thymine alkyltransferase]; [dCas9 or Cas9 nickase] -[optional linker sequence] -[iDAR] - [optional linker sequence] -[thymine alkyltransferase]; or [dCas9 or Cas9 nickase] -[optional linker sequence] -[thymine alkyltransferase] -[optional linker sequence] -[iDAR].

Reduced off-target effects

[0243] In some embodiments, the target nucleotide sequence is a DNA sequence in a genome, e.g. a eukaryotic genome. In certain embodiments, the target nucleotide sequence is in a mammalian (e.g. a human) genome. In certain embodiments, the target nucleotide sequence is in a human genome. In other embodiments, the target nucleotide sequence is in the genome of a rodent, such as a mouse or rate. In other embodiments, the target nucleotide sequence is in the genome of a domesticated animal, such as a horse, cat, dog, or rabbit.

[0244] Some embodiments of the disclosure are based on the recognition that any of the fusion proteins provided herein are capable of modifying a specific nucleobase without generating a significant proportion of indels. An“indel”, as used herein, refers to the insertion or deletion of a nucleobase within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate fusion proteins that efficiently modify (e.g. alkylate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the fusion proteins provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations) versus indels. [0245] In some embodiments, the fusion proteins provided herein are capable of generating a ratio of intended point mutations to indels that is greater than 1:1. In some embodiments, the fusion proteins provided herein are capable of generating a ratio of intended point mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method, for example the methods used in the below Examples. In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively.

[0246] In some embodiments, the fusion proteins provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a fusion protein or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a fusion protein. In some embodiments, any of the fusion proteins provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a fusion protein. In some embodiments, an number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a fusion protein.

[0247] Some embodiments of the disclosure are based on the recognition that any of the fusion proteins provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific fusion protein bound to a gRNA, specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation associated with a disease, disorder, or condition. In some embodiments, the intended mutation is the correction of a thymine (T) to adenine (A) point mutation associated with a disease, disorder, or condition. In some embodiments, the intended mutation is the correction of an adenine (A) to thymine (T) point mutation associated with a disease, disorder, or condition. In some embodiments, the intended mutation is the correction of a thymine (T) to adenine (A) point mutation within the coding region of a gene. In some embodiments, the intended mutation is the correction of an adenine (A) to thymine (T) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some embodiments, any of the fusion proteins provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point m utati o n s : u n i n t c n dcd point mutations) that is greater than 1: 1. In some embodiments, any of the fusion proteins provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point

m utati o n s : u n i n t c n dcd point mutations) that is at least 1.5: 1, at least 2: 1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, at least 4: 1, at least 4.5: 1, at least 5: 1, at least 5.5: 1, at least 6: 1, at least 6.5: 1, at least 7: 1, at least 7.5: 1, at least 8: 1, at least 10: 1, at least 12: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, at least 500: 1, or at least 1000: 1, or more.

[0248] Some embodiments of the disclosure are based on the recognition that the formation of indels in a region of a nucleic acid may be limited by nicking the non-edited strand opposite to the strand in which edits are introduced. This nick serves to direct mismatch repair machinery to the non-edited strand, ensuring that the chemically modified nucleobase is not interpreted as a lesion by the machinery. This nick may be created by the use of an nCas9. The methods provided in this disclosure comprise cutting (or nicking) the non-edited strand of the double-stranded DNA, for example, wherein the one strand comprises the A of the target T: A nucleobase pair, or the T of the T:A nucleobase pair. Guide sequences (e.g., guide RNAs)

[0249] The present disclosure further provides guide RNAs for use in accordance with the disclosed methods of editing. The disclosure provides guide RNAs that are designed to recognize target sequences. Such gRNAs may be designed to have guide sequences (or “spacers”) having complementarity to a protospacer within the target sequence. Guide RNAs are also provided for use with one or more of the disclosed fusion proteins, e.g., in the disclosed methods of editing a nucleic acid molecule. Such gRNAs may be designed to have guide sequences having complementarity to a protospacer within a target sequence to be edited, and to have backbone sequences that interact specifically with the napDNAbp domains of any of the disclosed base editors, such as Cas9 nickase domains of the disclosed base editors.

[0250] In various embodiments, the TABEs may be complexed, bound, or otherwise associated with (e.g., via any type of covalent or non-covalent bond) one or more guide sequences, i.e., the sequence which becomes associated or bound to the base editor and directs its localization to a specific target sequence having complementarity to the guide sequence or a portion thereof. The particular design embodiments of a guide sequence will depend upon the nucleotide sequence of a genomic target site of interest (i.e., the desired site to be edited) and the type of napDNAbp (e.g., type of Cas protein) present in the base editor, among other factors, such as PAM sequence locations, percent G/C content in the target sequence, the degree of microhomology regions, secondary structures, etc.

[0251] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%,

85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.

[0252] In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence- specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, the components of a base editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0253] A guide sequence may be selected to target any target sequence. In some

embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S.

pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 20) where

NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) (SEQ ID NO: 21) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 22) where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) (SEQ ID NO: 23) has a single occurrence in the genome. For the S. thermophilus CRISPRlCas9, a unique target sequence in a genome may include a Cas9 target site of the form

MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 24) where

NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) (SEQ ID NO: 25) has a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR 1 Cas9 target site of the form

MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 26) where

NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) (SEQ ID NO: 27) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form

MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 28) where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) (SEQ ID NO: 29) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 30) where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) (SEQ ID NO: 31) has a single occurrence in the genome. In each of these sequences“M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique.

[0254] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker & Stiegler {Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online Webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see, e.g., A. R. Gruber el al., 2008, Cell 106(1): 23-24; and PA Carr & GM Church, 2009, Nature Biotechnology 27(12): 1151- 62). Additional algorithms may be found in Chuai, G. el al, DeepCRISPR: optimized

CRISPR guide RNA design by deep learning , Genome Biol. 19:80 (2018), and U.S.

Application Ser. No. 61/836,080 and U.S. Patent No. 8,871,445, issued October 28, 2014, the entireties of each of which are incorporated herein by reference.

[0255] In general, a tracr mate sequence includes any sequence that has sufficient

complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a complex at a target sequence, wherein the complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the disclosure, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In certain embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the disclosure, the transcript has at most five hairpins.

In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. Further non limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5' to 3'), where“N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator:

(1) NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggctt

catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 36);

(2)

NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 37); (3)

NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaa atca acaccctgtcattttatggcagggtgtTTTTT (SEQ ID NO: 38); (4)

NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttga aaa agtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 39); (5)

NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaactt gaa aaagtgTTTTTTT (SEQ ID NO: 40); and (6)

NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTT TT TTT (SEQ ID NO: 41). In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.

[0256] It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and an alkyltransferase, as disclosed herein, to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the

Cas9:nucleic acid editing enzyme/domain fusion protein.

[0257] In some embodiments, the guide RNAs for use in accordance with the disclosed methods of editing comprise a backbone structure that is recognized by an S. pyogenes Cas9 protein or domain, such as an SpCas9 domain of the disclosed base editors. The backbone structure recognized by an SpCas9 protein may comprise the sequence 5'-[guide sequence]- guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuu uu-3' (SEQ ID NO: 42), wherein the guide sequence comprises a sequence that is complementary to the protospacer of the target sequence. See U.S. Publication No.

2015/0166981, published June 18, 2015, the disclosure of which is incorporated by reference herein. The guide sequence is typically 20 nucleotides long.

[0258] In other embodiments, the guide RNAs for use in accordance with the disclosed methods of editing comprise a backbone structure that is recognized by an S. aureus Cas9 protein. The backbone structure recognized by an SaCas9 protein may comprise the sequence 5 '-[guide sequence] - guuuuaguacucuguaaugaaaauuacagaaucuacuaaaacaaggcaaaaugccguguuuaucucgucaacuuguugg cgagauuuuuuu-3' (SEQ ID NO: 154).

The sequences of suitable guide RNAs for targeting the disclosed fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein.

Additional guide sequences are are well known in the art and can be used with the base editors described herein. Additional exemplary guide sequences are disclosed in, for example, Jinek M., et al., Science 337:816-821(2012); Mali P, Esvelt KM & Church GM (2013) Cas9 as a versatile tool for engineering biology, Nature Methods , 10, 957-963; Li JF et al, (2013) Multiplex and homologous recombination-mediated genome editing in

Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9, Nature Biotechnology , 31, 688-691; Hwang, W.Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system, Nature Biotechnology 31, 227-229 (2013); Cong L et al., (2013) Multiplex genome engineering using CRIPSR/Cas systems, Science, 339, 819-823; Cho SW et al., (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease, Nature Biotechnology, 31, 230-232; Jinek, M. et al., RNA-programmed genome editing in human cells, eLife 2, e00471 (2013); Dicarlo, J.E. el al., Genome engineering in

Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acid Res. (2013); Briner AE et al., (2014) Guide RNA functional modules direct Cas9 activity and orthogonality, Mol Cell, 56, 333-339, the entire contents of each of which are herein incorporated by reference.

Methods for making fusion proteins

[0259] The disclosure further relates in various aspects to methods of making the disclosed fusion proteins by various modes of manipulation that include, but are not limited to, codon optimization to achieve greater expression levels in a cell, and the use of nuclear localization sequences (NLSs), preferably at least two NLSs, e.g., two bipartite NLSs, to increase the localization of the expressed fusion proteins into a cell nucleus.

[0260] The fusion proteins contemplated herein can include modifications that result in increased expression, for example, through codon optimization.

[0261] In some embodiments, the fusion proteins (or a component thereof) is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including, but not limited to, human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The

predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the“Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0262] The above description is meant to be non-limiting with regard to making fusion proteins having increased expression, and thereby increase editing efficiencies.

Directed evolution methods (e.g., PACE or PANCE)

[0263] Various embodiments of the disclosure relate to providing directed evolution methods and systems (e.g., appropriate vectors, cells, phage, flow vessels, etc.) for engineering of the base editors or base editor domains of the present disclosure. The disclosure provides vector systems for the disclosed directed evolution methods to engineer any of the disclosed base editors or base editor fomains (e.g., the adenosine deaminase domains of any of the disclosed base editors).

[0264] The directed evolution vector systems and methods provided herein allow for a gene of interest (e.g., a base editor- or adenosine deaminase-encoding gene) in a viral vector to be evolved over multiple generations of viral life cycles in a flow of host cells to acquire a desired function or activity.

[0265] In PACE, the gene under selection is encoded on the M13 bacteriophage genome. Its activity is linked to M13 propagation by controlling expression of gene III so that only active variants produce infectious progeny phage. Phage are continuously propagated and mutagenized, but mutations accumulate only in the phage genome, not the host or its selection circuit, because fresh host cells are continually flowed into (and out of) the growth vessel, effectively resetting the selection background.

[0266] PACE enables the rapid continuous evolution of biomolecules through many generations of mutation, selection, and replication per day. During PACE, host E. coli cells continuously dilute a population of bacteriophage (selection phage, SP) containing the gene of interest. The gene of interest replaces gene III on the SP, which is required for progeny phage infectivity. SP containing desired gene variants trigger host-cell gene III expression from an accessory plasmid (AP). Host-cell DNA plasmids encode a genetic circuit that links the desired activity of the protein encoded in the SP to the expression of gene III on the AP. Thus, SP variants containing desired gene variants can propagate, while phage encoding inactive variants do not generate infectious progeny and are rapidly diluted out of the culture vessel (or lagoon). An arabinose-inducible mutagenesis plasmid (MP) controls the phage mutation rate.

[0267] The key to new PACE selections is linking gene III expression to the activity of interest. A low stringency selection was designed in which base editing activates T7 RNA polymerase, which transcribes gill. A single editing event can lead to high output

amplification immediately upon transcription of the edited DNA. Reference is made to International Patent Publication WO 2019/023680, published January 31, 2019; Badran, A.H. & Liu, D.R. In vivo continuous directed evolution. Curr. Opin. Chem. Biol. 24, 1-10 (2015); Dickinson, B.C., Packer, M.S., Badran, A.H. & Liu, D.R. A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations. Nat. Commun. 5, 5352 (2014); Hubbard, B.P. et al. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nat. Methods 12, 939-942 (2015); Wang, T., Badran, A.H., Huang, T.P. & Liu, D.R. Continuous directed evolution of proteins with improved soluble expression. Nat. Chem. Biol. 14, 972-980 (2018), and Thuronyi, B.W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol., 1070-1079 (2019), each of which is herein incorporated by reference. In some embodiments, the viral vector or the phage is a filamentous phage, for example, an M13 phage, such as an M13 selection phage as described in more detail elsewhere herein.

[0268] In some such embodiments, the gene required for the production of infectious viral particles is the M13 gene III (gill).

[0269] In some embodiments, the viral vector infects mammalian cells. In some

embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a vesicular stomatitis virus (VSV) vector. As a dsRNA virus, VSV has a high mutation rate, and can carry cargo, including a gene of interest, of up to 4.5 kb in length. The generation of infectious VSV particles requires the envelope protein VSV-G, a viral glycoprotein that mediates phosphatidylserine attachment and cell entry. VSV can infect a broad spectrum of host cells, including mammalian and insect cells. VSV is therefore a highly suitable vector for continuous evolution in human, mouse, or insect host cells. Similarly, other retroviral vectors that can be pseudotyped with VSV-G envelope protein are equally suitable for continuous evolution processes as described herein.

[0270] It is known to those of skill in the art that many retroviral vectors, for example, Murine Leukemia Vims vectors, or Lentiviral vectors can efficiently be packaged with VSV- G envelope protein as a substitute for the vims’s native envelope protein. In some embodiments, such VSV-G packagable vectors are adapted for use in a continuous evolution system in that the native envelope (env) protein (e.g., VSV-G in VSVS vectors, or env in MLV vectors) is deleted from the viral genome, and a gene of interest is inserted into the viral genome under the control of a promoter that is active in the desired host cells. The host cells, in turn, express the VSV-G protein, another env protein suitable for vector

pseudotyping, or the viral vector’s native env protein, under the control of a promoter the activity of which is dependent on an activity of a product encoded by the gene of interest, so that a viral vector with a mutation leading to increased activity of the gene of interest will be packaged with higher efficiency than a vector with baseline or a loss-of-function mutation.

[0271] In some embodiments, mammalian host cells are subjected to infection by a continuously evolving population of viral vectors, for example, VSV vectors comprising a gene of interest and lacking the VSV-G encoding gene, wherein the host cells comprise a gene encoding the VSV-G protein under the control of a conditional promoter. Such retrovirus-bases system could be a two-vector system (the viral vector and an expression construct comprising a gene encoding the envelope protein), or, alternatively, a helper virus can be employed, for example, a VSV helper vims. A helper virus typically comprises a truncated viral genome deficient of structural elements required to package the genome into viral particles, but including viral genes encoding proteins required for viral genome processing in the host cell, and for the generation of viral particles. In such embodiments, the viral vector-based system could be a three-vector system (the viral vector, the expression construct comprising the envelope protein driven by a conditional promoter, and the helper vims comprising viral functions required for viral genome propagation but not the envelope protein). In some embodiments, expression of the five genes of the VSV genome from a helper vims or expression constmct in the host cells, allows for production of infectious viral particles carrying a gene of interest, indicating that unbalanced gene expression permits viral replication at a reduced rate, suggesting that reduced expression of VSV-G would indeed serve as a limiting step in efficient viral production.

[0272] One advantage of using a helper vims is that the viral vector can be deficient in genes encoding proteins or other functions provided by the helper vims, and can, accordingly, carry a longer gene of interest. In some embodiments, the helper vims does not express an envelope protein, because expression of a viral envelope protein is known to reduce the infectability of host cells by some viral vectors via receptor interference. Viral vectors, for example retroviral vectors, suitable for continuous evolution processes, their respective envelope proteins, and helper vimses for such vectors, are well known to those of skill in the art. For an overview of some exemplary viral genomes, helper vimses, host cells, and envelope proteins suitable for continuous evolution procedures as described herein, see Coffin et al., Retroviruses, CSHL Press 1997, ISBN0-87969-571-4, incorporated herein in its entirety.

[0273] In some embodiments, the incubating of the host cells is for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles. In certain embodiments, the viral vector is an M13 phage, and the length of a single viral life cycle is about 10-20 minutes.

[0274] In some embodiments, a viral vector/host cell combination is chosen in which the life cycle of the viral vector is significantly shorter than the average time between cell divisions of the host cell. Average cell division times and viral vector life cycle times are well known in the art for many cell types and vectors, allowing those of skill in the art to ascertain such host cell/vector combinations. In certain embodiments, host cells are being removed from the population of host cells contacted with the viral vector at a rate that results in the average time of a host cell remaining in the host cell population before being removed to be shorter than the average time between cell divisions of the host cells, but to be longer than the average life cycle of the viral vector employed. The result of this is that the host cells, on average, do not have sufficient time to proliferate during their time in the host cell population while the viral vectors do have sufficient time to infect a host cell, replicate in the host cell, and generate new viral particles during the time a host cell remains in the cell population.

This assures that the only replicating nucleic acid in the host cell population is the viral vector, and that the host cell genome, the accessory plasmid, or any other nucleic acid constructs cannot acquire mutations allowing for escape from the selective pressure imposed.

[0275] For example, in some embodiments, the average time a host cell remains in the host cell population is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes.

[0276] In some embodiments, the average time a host cell remains in the host cell population depends on how fast the host cells divide and how long infection (or conjugation) requires.

In general, the flow rate should be faster than the average time required for cell division, but slow enough to allow viral (or conjugative) propagation. The former will vary, for example, with the media type, and can be delayed by adding cell division inhibitor antibiotics (FtsZ inhibitors in E. coli, etc.). Since the limiting step in continuous evolution is production of the protein required for gene transfer from cell to cell, the flow rate at which the vector washes out will depend on the current activity of the gene(s) of interest. In some embodiments, titratable production of the protein required for the generation of infectious particles, as described herein, can mitigate this problem. In some embodiments, an indicator of phage infection allows computer-controlled optimization of the flow rate for the current activity level in real-time.

[0277] In some embodiments, the fresh host cells comprise the accessory plasmid required for selection of viral vectors, for example, the accessory plasmid comprising the gene required for the generation of infectious phage particles that is lacking from the phages being evolved. In some embodiments, the host cells are generated by contacting an uninfected host cell with the relevant vectors, for example, the accessory plasmid and, optionally, a mutagenesis plasmid, and growing an amount of host cells sufficient for the replenishment of the host cell population in a continuous evolution experiment. Methods for the introduction of plasmids and other gene constructs into host cells are well known to those of skill in the art and the disclosure is not limited in this respect. For bacterial host cells, such methods include, but are not limited to, electroporation and heat-shock of competent cells.

[0278] In some embodiments, the accessory plasmid comprises a selection marker, for example, an antibiotic resistance marker, and the fresh host cells are grown in the presence of the respective antibiotic to ensure the presence of the plasmid in the host cells. Where multiple plasmids are present, different markers are typically used. Such selection markers and their use in cell culture are known to those of skill in the art, and the disclosure is not limited in this respect.

[0279] In some embodiments, the selection marker is a spectinomycin antibiotic resistance marker. Cells are transformed with a selection plasmid containing an inactivated

spectinomycin resistance gene with a premature stop codon or a mutation at an active site (K205T or D182V) that each requires T:A to A:T editing to correct. Cells that fail to install the correct transversion mutation in the spectinomycin resistance gene will die, while cells that make the correction will survive. E. coli cells expressing an sgRNA targeting the K205T or D182V defect in the spectinomycin resistance gene and a nucleobase modification domain-dCas9 fusion protein were plated onto 2xYT agar with 256 pg/mL of spectinomycin. Surviving colonies (measured through CFUs) were sequenced to find consensus mutations in the fusion proteins expressed in the evolved survivors. A similar selection assay was used to evolve adenosine deaminase activity in DNA during adenine base editor development, as described in Gaudelli, N. M. el al, Programmable base editing of A·T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017), herein incorporated in its entirety by reference.

[0280] In some embodiments, the selection marker is a chloramphenicol antibiotic resistance marker. Cells are transformed with a selection plasmid containing an inactivated

chloramphenicol resistance gene with a mutation at an active site (H193Q) that requires T:A to A:T editing to correct. Cells that fail to install the correct transversion mutation in the chloramphenicol resistance gene will die, while cells that make the correction will survive.

E. coli cells expressing an sgRNA targeting the H193Q defect in the chloramphenicol resistance gene and a nucleobase modification domain-dCas9 fusion protein were plated onto 2xYT agar with 256 pg/mL of chloramphenicol. Surviving colonies (measured through CFUs) were sequenced to find consensus mutations in the fusion proteins expressed in the evolved survivors.

[0281] In other embodiments, the selection marker is a carbenicillin antibiotic resistance marker. Cells are transformed with a selection plasmid containing an inactivated

carbenicillin resistance gene with a premature stop codon (Y95X) or a mutation at an active site (S233A or E166A) that each require T:A to A:T editing to correct (FIGs. 1A-1B). Cells that fail to install the correct transversion mutation in the carbenicillin resistance gene will die, while cells that make the correction will survive. E. coli cells expressing an sgRNA targeting the defect in the carbenecillin resistance gene and a nucleobase modification domain-dCas9 fusion protein were plated onto 2xYT agar with 256 pg/mL of carbenicillin. Surviving colonies (measured through CFUs) were sequenced to find consensus mutations in the fusion proteins expressed in the evolved survivors.

[0282] In some embodiments, the host cell population in a continuous evolution experiment is replenished with fresh host cells growing in a parallel, continuous culture. In some embodiments, the cell density of the host cells in the host cell population contacted with the viral vector and the density of the fresh host cell population is substantially the same.

[0283] Typically, the cells being removed from the cell population contacted with the viral vector comprise cells that are infected with the viral vector and uninfected cells. In some embodiments, cells are being removed from the cell populations continuously, for example, by effecting a continuous outflow of the cells from the population. In other embodiments, cells are removed semi-continuously or intermittently from the population. In some embodiments, the replenishment of fresh cells will match the mode of removal of cells from the cell population, for example, if cells are continuously removed, fresh cells will be continuously introduced. However, in some embodiments, the modes of replenishment and removal may be mismatched, for example, a cell population may be continuously replenished with fresh cells, and cells may be removed semi-continuously or in batches.

[0284] In some embodiments, the rate of fresh host cell replenishment and/or the rate of host cell removal is adjusted based on quantifying the host cells in the cell population. For example, in some embodiments, the turbidity of culture media comprising the host cell population is monitored and, if the turbidity falls below a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect an increase in the number of host cells in the population, as manifested by increased cell culture turbidity. In other embodiments, if the turbidity rises above a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect a decrease in the number of host cells in the population, as manifested by decreased cell culture turbidity. Maintaining the density of host cells in the host cell population within a specific density range ensures that enough host cells are available as hosts for the evolving viral vector population, and avoids the depletion of nutrients at the cost of viral packaging and the accumulation of cell-originated toxins from overcrowding the culture.

[0285] In some embodiments, the cell density in the host cell population and/or the fresh host cell density in the inflow is about 102 cells/ml to about 1012 cells/ml. In some embodiments, the host cell density is about 102 cells/ml, about 103 cells/ml, about 104 cells/ml, about 105 cells/ml, about 5- 105 cells/ml, about 106 cells/ml, about 5- 106 cells/ml, about 107 cells/ml, about 5- 107 cells/ml, about 108 cells/ml, about 5- 108 cells/ml, about 109 cells/ml, about 5- 109 cells/ml, about 1010 cells/ml, or about 5- 1010 cells/ml. In some embodiments, the host cell density is more than about 1010 cells/ml.

[0286] In some embodiments, the host cell population is contacted with a mutagen. In some embodiments, the cell population contacted with the viral vector (e.g., the phage), is continuously exposed to the mutagen at a concentration that allows for an increased mutation rate of the gene of interest, but is not significantly toxic for the host cells during their exposure to the mutagen while in the host cell population. In other embodiments, the host cell population is contacted with the mutagen intermittently, creating phases of increased mutagenesis, and accordingly, of increased viral vector diversification. For example, in some embodiments, the host cells are exposed to a concentration of mutagen sufficient to generate an increased rate of mutagenesis in the gene of interest for about 10%, about 20%, about 50%, or about 75% of the time. [0287] In some embodiments, the host cells comprise a mutagenesis expression construct, for example, in the case of bacterial host cells, a mutagenesis plasmid. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding a mutagenesis- promoting gene product, for example, a proofreading-impaired DNA polymerase. In other embodiments, the mutagenesis plasmid, including a gene involved in the SOS stress response, (e.g., UmuC, UmuD', and/or RecA). In some embodiments, the mutagenesis- promoting gene is under the control of an inducible promoter. Suitable inducible promoters are well known to those of skill in the art and include, for example, arabinose-inducible promoters, tetracycline or doxycyclin-inducible promoters, and tamoxifen-inducible promoters. In some embodiments, the host cell population is contacted with an inducer of the inducible promoter in an amount sufficient to effect an increased rate of mutagenesis. For example, in some embodiments, a bacterial host cell population is provided in which the host cells comprise a mutagenesis plasmid in which a dnaQ926, UmuC, UmuD', and RecA expression cassette is controlled by an arabinose-inducible promoter. In some such embodiments, the population of host cells is contacted with the inducer, for example, arabinose in an amount sufficient to induce an increased rate of mutation.

[0288] In some embodiments, diversifying the viral vector population is achieved by providing a flow of host cells that does not select for gain-of-function mutations in the gene of interest for replication, mutagenesis, and propagation of the population of viral vectors. In some embodiments, the host cells are host cells that express all genes required for the generation of infectious viral particles, for example, bacterial cells that express a complete helper phage, and, thus, do not impose selective pressure on the gene of interest. In other embodiments, the host cells comprise an accessory plasmid comprising a conditional promoter with a baseline activity sufficient to support viral vector propagation even in the absence of significant gain-of-function mutations of the gene of interest. This can be achieved by using a“leaky” conditional promoter, by using a high-copy number accessory plasmid, thus amplifying baseline leakiness, and/or by using a conditional promoter on which the initial version of the gene of interest effects a low level of activity while a desired gain- of-function mutation effects a significantly higher activity.

[0289] Detailed methods of procedures for directing continuous evolution of base editors in a population of host cells using phage particles are disclosed in International PCT Application, PCT/US 2009/056194, filed September 8, 2009, published as WO 2010/028347 on March 11, 2010; International PCT Application, PCT/US2011/066747, filed December 22, 2011, published as WO 2012/088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application, PCT/US2015/012022, filed January 20, 2015, published as WO 2015/134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued January 15, 2019; International Application No. PCT/US2019/37216, published as WO 2019/241649 on December 19, 2019, International Patent Publication WO 2019/023680, published January 31, 2019, International PCT Application, PCT/US2016/027795, filed April 15, 2016, published as WO 2016/168631 on October 20, 2016, and International Application No. PCT/US2019/47996, filed August 23, 2019, each of which are incorporated herein by reference.

[0290] Methods and strategies to design conditional promoters suitable for carrying out the selection strategies described herein are well known to those of skill in the art. For an overview over exemplary suitable selection strategies and methods for designing conditional promoters driving the expression of a gene required for cell-cell gene transfer, e.g., gene III (gill), see Vidal and Legrain, Yeast n-hybrid review, Nucleic Acid Res. 27, 919 (1999), incorporated herein in its entirety.

[0291] The disclosure provides vectors for the continuous evolution processes. In some embodiments, phage vectors for phage-assisted continuous evolution are provided. In some embodiments, a selection phage is provided that comprises a phage genome deficient in at least one gene required for the generation of infectious phage particles and a gene of interest to be evolved. Reference is made to International Patent Publication WO 2019/023680, published January 31, 2019, herein incorporated by reference.

[0292]

[0293] For example, in some embodiments, a population of host cells comprising a high-copy accessory plasmid with a gene required for the generation of infectious phage particles is contacted with a selection phage comprising a gene of interest, wherein the accessory plasmid comprises a conditional promoter driving expression of the gene required for the generation from a conditional promoter, the activity of which is dependent on the activity of a gene product encoded by the gene of interest. In some such embodiments, a low stringency selection phase can be achieved by designing the conditional promoter in a way that the initial gene of interest exhibits some activity on that promoter. For example, if a

transcriptional activator, such as a T7RNAP or a transcription factor is to be evolved to recognize a non-native target DNA sequence (e.g., a T3RNAP promoter sequence, on which T7RNAP has no activity), a low-stringency accessory plasmid can be designed to comprise a conditional promoter in which the target sequence comprises a desired characteristic, but also retains a feature of the native recognition sequence that allows the transcriptional activator to recognize the target sequence, albeit with less efficiency than its native target sequence.

Initial exposure to such a low-stringency accessory plasmid comprising a hybrid target sequence (e.g., a T7/T3 hybrid promoter, with some features of the ultimately desired target sequence and some of the native target sequence) allows the population of phage vectors to diversify by acquiring a plurality of mutations that are not immediately selected against based on the permissive character of the accessory plasmid. Such a diversified population of phage vectors can then be exposed to a stringent selection accessory plasmid, for example, a plasmid comprising in its conditional promoter the ultimately desired target sequence that does not retain a feature of the native target sequence, thus generating a strong negative selective pressure against phage vectors that have not acquired a mutation allowing for recognition of the desired target sequence.

[0294] In some embodiments, an initial host cell population contacted with a population of evolving viral vectors is replenished with fresh host cells that are different from the host cells in the initial population. For example, in some embodiments, the initial host cell population is made of host cells comprising a low-stringency accessory plasmid, or no such plasmid at all, or are permissible for viral infection and propagation. In some embodiments, after diversifying the population of viral vectors in the low-stringency or no-selection host cell population, fresh host cells are introduced into the host cell population that impose a more stringent selective pressure for the desired function of the gene of interest. For example, in some embodiments, the secondary fresh host cells are not permissible for viral replication and propagation anymore. In some embodiments, the stringently selective host cells comprise an accessory plasmid in which the conditional promoter exhibits none or only minimal baseline activity, and/or which is only present in low or very low copy numbers in the host cells.

[0295] Such methods involving host cells of varying selective stringency allow for harnessing the power of continuous evolution methods as provided herein for the evolution of functions that are completely absent in the initial version of the gene of interest, for example, for the evolution of a transcription factor recognizing a foreign target sequence that a native transcription factor, used as the initial gene of interest, does not recognize at all. Or, for another example, the recognition of a desired target sequence by a DNA-binding protein, a recombinase, a nuclease, a zinc-finger protein, or an RNA-polymerase, that does not bind to or does not exhibit any activity directed towards the desired target sequence.

[0296] In some embodiments, negative selection is applied during a continuous evolution method as described herein, by penalizing undesired activities. In some embodiments, this is achieved by causing the undesired activity to interfere with pill production. For example, expression of an antisense RNA complementary to the gill RBS and/or start codon is one way of applying negative selection, while expressing a protease (e.g., TEV) and engineering the protease recognition sites into pill is another.

[0297] In some embodiments, negative selection is applied during a continuous evolution method as described herein, by penalizing the undesired activities of evolved products. This is useful, for example, if the desired evolved product is an enzyme with high specificity, for example, a transcription factor or protease with altered, but not broadened, specificity. In some embodiments, negative selection of an undesired activity is achieved by causing the undesired activity to interfere with pill production, thus inhibiting the propagation of phage genomes encoding gene products with an undesired activity. In some embodiments, expression of a dominant-negative version of pill or expression of an antisense RNA complementary to the gill RBS and/or gill start codon is linked to the presence of an undesired activity. In some embodiments, a nuclease or protease cleavage site, the recognition or cleavage of which is undesired, is inserted into a pill transcript sequence or a pill amino acid sequence, respectively. In some embodiments, a transcriptional or translational repressor is used that represses expression of a dominant negative variant of pill and comprises a protease cleavage site the recognition or cleaveage of which is undesired.

[0298] In some embodiments, counter- selection against activity on non-target substrates is achieved by linking undesired evolved product activities to the inhibition of phage propagation. For example, in some embodiments, in which a transcription factor is evolved to recognize a specific target sequence, but not an undesired off-target sequence, a negative selection cassette is employed, comprising a nucleic acid sequence encoding a dominant negative version of pill (plll-neg) under the control of a promoter comprising the off-target sequence. If an evolution product recognizes the off-target sequence, the resulting phage particles will incorporate plll-neg, which results in an inhibition of phage infective potency and phage propagation, thus constituting a selective disadvantage for any phage genomes encoding an evolution product exhibiting the undesired, off-target activity, as compared to evolved products not exhibiting such an activity. In some embodiments, a dual selection strategy is applied during a continuous evolution experiment, in which both positive selection and negative selection constructs are present in the host cells. In some such embodiments, the positive and negative selection constructs are situated on the same plasmid, also referred to as a dual selection accessory plasmid. [0299] For example, in some embodiments, a dual selection accessory plasmid is employed comprising a positive selection cassette, comprising a pill-encoding sequence under the control of a promoter comprising a target nucleic acid sequence, and a negative selection cassette, comprising a plll-neg encoding cassette under the control of a promoter comprising an off-target nucleic acid sequence. One advantage of using a simultaneous dual selection strategy is that the selection stringency can be fine-tuned based on the activity or expression level of the negative selection construct as compared to the positive selection construct.

Another advantage of a dual selection strategy is the selection is not dependent on the presence or the absence of a desired or an undesired activity, but on the ratio of desired and undesired activities, and, thus, the resulting ratio of pill and plll-neg that is incorporated into the respective phage particle.

[0300] Some embodiments of this disclosure provide or utilize a dominant negative variant of pill (plll-neg). These embodiments are based on the discovery that a pill variant that comprises the two N-terminal domains of pill and a truncated, termination-incompetent C- terminal domain is not only inactive but is a dominant-negative variant of pill. A pill variant comprising the two N-terminal domains of pill and a truncated, termination-incompetent C- terminal domain was described in Bennett, N. J.; Rakonjac, J., Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pill. Journal of

Molecular Biology 2006, 356 (2), 266-73; the entire contents of which are incorporated herein by reference.

[0301] Positive and negative selection strategies can further be designed to link non-DNA directed activities to phage propagation efficiency. For example, protease activity towards a desired target protease cleavage site can be linked to pill expression by devising a repressor of gene expression that can be inactivated by a protease recognizing the target site. In some embodiments, pill expression is driven by a promoter comprising a binding site for such a repressor. Suitable transcriptional repressors are known to those in the art, and one exemplary repressor is the lambda repressor protein, that efficiently represses the lambda promoter pR and can be modified to include a desired protease cleavage site (see, e.g., Sices, H. J.; Kristie, T. M., A genetic screen for the isolation and characterization of site-specific proteases. Proc. Natl. Acad. Sci. USA 1998, 95 (6), 2828-33; and Sices, H. J. et al., Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease. AIDS Res Hum Retroviruses 2001, 17 (13), 1249-55, the entire contents of each of which are incorporated herein by reference). The lambda repressor (cl) contains an N-terminal DNA binding domain and a C-terminal dimerization domain. These two domains are connected by a flexible linker. Efficient transcriptional repression requires the dimerization of cl, and, thus, cleavage of the linker connecting dimerization and binding domains results in abolishing the repressor activity of cl.

[0302] Some embodiments provide a pill expression construct that comprises a pR promoter (containing cl binding sites) driving expression of pill. When expressed together with a modified cl comprising a desired protease cleavage site in the linker sequence connecting dimerization and binding domains, the cl molecules will repress pill transcription in the absence of the desired protease activity, and this repression will be abolished in the presence of such activity, thus providing a linkage between protease cleavage activity and an increase in pill expression that is useful for positive PACE protease selection. Some embodiments provide a negative selection strategy against undesired protease activity in PACE evolution products. In some embodiments, the negative selection is conferred by an expression cassette comprising a plll-neg encoding nucleic acid under the control of a cl-repressed promoter. When co-expressed with a cl repressor protein comprising an undesired protease cleavage site, expression of plll-neg will occur in cell harboring phage expressing a protease exhibiting protease activity towards the undesired target site, thus negatively selecting against phage encoding such undesired evolved products. A dual selection for protease target specificity can be achieved by co-expressing cl-repressible pill and plll-neg encoding expression constructs with orthogonal cl variants recognizing different nucleic acid target sequences, and thus allowing for simultaneous expression without interfering with each other. Orthogonal cl variants in both dimerization specificity and DNA-binding specificity are known to those of skill in the art (see, e.g., Wharton, R. P.; Ptashne, M., Changing the binding specificity of a repressor by redesigning an alphahelix. Nature 1985, 316 (6029), 601-5; and Wharton, R. P.; Ptashne, M., A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact. Nature 1987, 326 (6116), 888-91, the entire contents of each of which are incorporated herein by reference).

[0303] Other selection schemes for gene products having a desired activity are well known to those of skill in the art or will be apparent from the instant disclosure. Selection strategies that can be used in continuous evolution processes and methods as provided herein include, but are not limited to, selection strategies useful in two-hybrid screens. For example, in the T7 RNAP selection strategy, successful base editing leads to a translation of T7 RNAP without a C-terminal proteolytic degaradation tag, which enables transcripton of genelll (or a luciferase reporter) from a T7 promoter. [0304] Two-hybrid accessory plasmid setups further permit the evolution of protein-protein interactions, and accessory plasmids requiring site-specific recombinase activity for production of the protein required for the generation of infectious viral particles, for example, pin, allow recombinases to be evolved to recognize any desired target site. A two-hybrid setup or a related one-hybrid setup can further be used to evolve DNA-binding proteins, while a three -hybrid setup can evolve RNA-protein interactions.

[0305] Biosynthetic pathways producing small molecules can also be evolved with a promoter or riboswitch (e.g., controlling gene III expression/translation) that is responsive to the presence of the desired small molecule. For example, a promoter that is transcribed only in the presence of butanol could be placed on the accessory plasmid upstream of gene III to optimize a biosynthetic pathway encoding the enzymes for butanol synthesis. A phage vector carrying a gene of interest that has acquired an activity boosting butanol synthesis would have a selective advantage over other phages in an evolving phage population that have not acquired such a gain-of-function. Alternatively, a chemical complementation system, for example, as described in Baker and Cornish, PNAS (2002), incorporated herein by reference, can be used to evolve individual proteins or enzymes capable of bond formation reactions. In other embodiments, a trans-splicing intron designed to splice itself into a particular target sequence can be evolved by expressing only the latter half of gene III from the accessory plasmid, preceded by the target sequence, and placing the other half (fused to the trans splicing intron) on the selection phage. Successful splicing would reconstitute full-length pill-encoding mRNA. Protease specificity and activity can be evolved by expressing pill fused to a large protein from the accessory plasmid, separated by a linker containing the desired protease recognition site. Cleavage of the linker by active protease encoded by the selection phage would result in infectious pill, while uncleaved pill would be unable to bind due to the blocking protein. Further, as described, for example, by Malmborg and

Borrebaeck 1997, a target antigen can be fused to the F pilus of a bacteria, blocking wild-type pill from binding. Phage displaying antibodies specific to the antigen could bind and infect, yielding enrichments of >1000-fold in phage display. In some embodiments, this system can be adapted for continuous evolution, in that the accessory plasmid is designed to produce wild-type pill to contact the tolA receptor and perform the actual infection (as the antibody- pIII fusion binds well but infects with low efficiency), while the selection phage encodes the pill-antibody fusion protein. Progeny phage containing both types of pill tightly adsorb to the F pilus through the antibody-antigen interaction, with the wild-type pill contacting tolA and mediating high-efficiency infection. To allow propagation when the initial antibody- antigen interaction is weak, a mixture of host cells could flow into the lagoon: a small fraction expressing wild-type pili and serving as a reservoir of infected cells capable of propagating any selection phage regardless of activity, while the majority of cells requires a successful interaction, serving as the“reward” for any mutants that improve their binding affinity. This last system, in some embodiments, can evolve new antibodies that are effective against a target pathogen faster than the pathogen itself can evolve, since the evolution rates of PACE and other systems described herein are higher than those of human- specific pathogens, for example, those of human viruses.

[0306] Methods and strategies to design conditional promoters suitable for carrying out the selections strategies described herein are well known to those of skill in the art. For an overview over exemplary suitable selection strategies and methods for designing conditional promoters driving the expression of a gene required for cell-cell gene transfer, e.g. gill, see Vidal and Legrain, Yeast n-hybrid review, Nucleic Acid Res. 27, 919 (1999), incorporated herein in its entirety.

[0307] By contrast, the PANCE method begins by first growing the host strain containing a mutagenesis plasmid of E. coli until optical density reaches Aeon = 0.3-0.5 in a large volume. The cells are re-transformed with the mutagenesis plasmid regularly to ensure the plasmid has not been inactivated. An aliquot of a desired concentration, often 2 mL, is then transferred to a smaller flask, supplemeted with inducing agent arabinose (Ara) for the mutagenesis plasmid, and infected with the selection phage (SP). To increase the titer level, a drift plasmid can also be provided that enables phage to propagate without passing the selection. Expression is under the control of an inducible promoter and can be turned on with 50 ng/mL of anhydrotetracycline. This culture is incubated at 37 °C for 8-12 h to facilitate phage growth, which is confirmed by determination of the phage titer. Following phage growth, an aliquot of infected cells is used to transfect a subsequent flask containing host E. coli. This process is continued until the desired phenotype is evolved for as many transfers as required, while increasing the stringency in stepwise fashion by decreasing the incubation time or titer of phage with which the bacteria is infected. Reference is made to Suzuki T. et al, Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13(12): 1261-1266 (2017), incorporated herein in its entirety.

[0308] Other non-continuous selection schemes for gene products having a desired activity are well known to those of skill in the art or will be apparent from the instant disclosure. In certain embodiments, following the successful directed evolution of one or more components of the transversion base editor (e.g., a Cas9 domain or a thymine alkyltransferase domain), methods of making the base editors comprise recombinant protein expression methodologies known to one of ordinary skill in the art.

Vectors

[0309] Several embodiments of the making and using the base editors of the disclosure relate to vector systems comprising one or more vectors encoding the disclosed TABEs. Vectors can be designed to clone and/or express the base editors of the disclosure. Vectors may also be designed to transfect the base editors of the disclosure into one or more cells, e.g., a target diseased eukaryotic cell for treatment with the base editor systems and methods disclosed herein.

[0310] Vectors may be designed for expression of base editor transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, base editor transcripts may be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovims expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods In Enzymology 185, Academic Press. San Diego, Calif. (1990). Alternatively, expression vectors encoding one or more base editors described herein may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0311] Vectors for rational mutagenesis methods such as PACE may be introduced and propagated in a prokaryotic cells. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.

[0312] Fusion expression vectors also may be used to express the base editors of the disclosure. Such vectors generally add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of a recombinant protein; (ii) to increase the solubility of a recombinant protein; and (iii) to aid in the purification of a recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion domain and the recombinant protein to enable separation of the recombinant protein from the fusion domain subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0313] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0314] In some embodiments, a vector is a yeast expression vector for expressing the base editors described herein. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0315] In some embodiments, a vector drives protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0316] In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2,

cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0317] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver- specific; Pinkert, el al., 1987. Genes Dev. 1: 268-277), lymphoid- specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron- specific promoters (e.g., the neurofilament promoter; Byme and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland- specific promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

[0318] The disclosure provides viral vectors for the continuous evolution processes. In some embodiments, phage vectors for phage-assisted continuous evolution are provided. In some embodiments, a selection phage is provided that comprises a phage genome deficient in at least one gene required for the generation of infectious phage particles and a gene of interest to be evolved.

[0319] For example, in some embodiments, the selection phage comprises an M13 phage genome deficient in a gene required for the generation of infectious M13 phage particles, for example, a full-length gill. In some embodiments, the selection phage comprises a phage genome providing all other phage functions required for the phage life cycle except the gene required for generation of infectious phage particles. In some such embodiments, an M13 selection phage is provided that comprises a gl, gll, gIV, gV, gVI, gVII, gVIII, glX, and a gX gene, but not a full-length gill. In some embodiments, the selection phage comprises a 3'- fragment of gill, but no full-length gill. The 3 '-end of gill comprises a promoter, and retaining this promoter activity is beneficial, in some embodiments, for an increased expression of gVI, which is immediately downstream of the gill 3 '-promoter, or a more balanced (wild-type phage-like) ratio of expression levels of the phage genes in the host cell, which, in turn, can lead to more efficient phage production. In some embodiments, the 3'- fragment of gill gene comprises the 3 '-gill promoter sequence. In some embodiments, the 3'- fragment of gill comprises the last 180 bp, the last 150 bp, the last 125 bp, the last 100 bp, the last 50 bp, or the last 25 bp of gill. In some embodiments, the 3'- fragment of gill comprises the last 180 bp of gill.

[0320] M13 selection phage is provided that comprises a gene of interest in the phage genome, for example, inserted downstream of the gVIII 3 '-terminator and upstream of the gIII-3 '-promoter. In some embodiments, an M13 selection phage is provided that comprises a multiple cloning site for cloning a gene of interest into the phage genome, for example, a multiple cloning site (MCS) inserted downstream of the gVIII 3 '-terminator and upstream of the gill- 3 '-promoter.

[0321] Some embodiments of this disclosure provide a vector system for continuous evolution procedures, comprising of a viral vector, for example, a selection phage, and a matching accessory plasmid. In some embodiments, a vector system for phage-based continuous directed evolution is provided that comprises (a) a selection phage comprising a gene of interest to be evolved, wherein the phage genome is deficient in a gene required to generate infectious phage; and (b) an accessory plasmid comprising the gene required to generate infectious phage particle under the control of a conditional promoter, wherein the conditional promoter is activated by a function of a gene product encoded by the gene of interest.

[0322] In some embodiments, the selection phage is an M 13 phage as described herein. For example, in some embodiments, the selection phage comprises an M13 genome including all genes required for the generation of phage particles, for example, gl, gll, gIV, gV, gVI, gVII, gVIII, glX, and gX gene, but not a full-length gill gene. In some embodiments, the selection phage genome comprises an FI or an M 13 origin of replication. In some embodiments, the selection phage genome comprises a 3 '-fragment of gill gene. In some embodiments, the selection phage comprises a multiple cloning site upstream of the gill 3 '-promoter and downstream of the gVIII 3 '-terminator.

[0323] In some embodiments, the selection phage does not comprise a full length gVI. GVI is similarly required for infection as gill and, thus, can be used in a similar fashion for selection as described for gill herein. However, it was found that continuous expression of pill renders some host cells resistant to infection by M13. Accordingly, it is desirable that pill is produced only after infection. This can be achieved by providing a gene encoding pill under the control of an inducible promoter, for example, an arabinose-inducible promoter as described herein, and providing the inducer in the lagoon, where infection takes place, but not in the turbidostat, or otherwise before infection takes place. In some embodiments, multiple genes required for the generation of infectious phage are removed from the selection phage genome, for example, gill and gVI, and provided by the host cell, for example, in an accessory plasmid as described herein.

[0324] The vector system may further comprise a helper phage, wherein the selection phage does not comprise all genes required for the generation of phage particles, and wherein the helper phage complements the genome of the selection phage, so that the helper phage genome and the selection phage genome together comprise at least one functional copy of all genes required for the generation of phage particles, but are deficient in at least one gene required for the generation of infectious phage particles.

[0325] In some embodiments, the accessory plasmid of the vector system comprises an expression cassette comprising the gene required for the generation of infectious phage under the control of a conditional promoter. In some embodiments, the accessory plasmid of the vector system comprises a gene encoding pill under the control of a conditional promoter the activity of which is dependent on a function of a product of the gene of interest.

[0326] In some embodiments, the vector system further comprises a mutagenesis plasmid, for example, an arabinose-inducible mutagenesis plasmid as described herein.

[0327] In some embodiments, the vector system further comprises a helper plasmid providing expression constructs of any phage gene not comprised in the phage genome of the selection phage or in the accessory plasmid.

[0328] In various embodiments of the vectors used herein in the continuous evolution processes may include the following component:

[0329] T7 RNA Polymerase

[0330] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRK MFERQLKAGE V ADN A A AKPLITTLLPKMIARIND WFEE VKAKRGKRPT AF QFLQEIK PE A V A YITIKTTLACLT S ADNTT V Q A V AS AIGR AIEDE ARF GRIRDLKAKHFKKN VEE QLNKRV GHVYKKAFMQVVE ADMLS KGLLGGE AW S S WHKEDSIH V GVRCIEMLIES TGM V S LHRQN AG V V GQDS ETIELAPE Y AE AIATRAG ALAGIS PMF QPC V VPPKPWTG IT GGG YW AN GRRPLALVRTHS KK ALMRYED V YMPE V YKAINIAQNT A WKINKKVL AVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKAR KSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTL AKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWW AEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAV NLLPS ET V QDIY GIV AKKVNEILQ AD AIN GTDNE V VT VTDENT GEIS EKVKLGTKALA GQWLA Y G VTRS VTKRS VMTLA Y GS KEF GFRQQ VLEDTIQP AIDS GKGLMFTQPN Q A AG YM AKLIWES V S VT V V A A VE AMNWLKS A AKLLA AE VKDKKT GEILRKRC A VHW VTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHS QDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVL ADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA* (SEQ ID NO: 50) [0331] Methods of Editing A Target Nucleobase Pair, Methods of Treatment, and Uses for the TABEsSome embodiments of the disclosure provide methods for editing a nucleic acid using the base editors described herein to effectuate substitution of an T:A base pair to an A:T base pair. In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double- stranded DNA sequence) with a complex comprising a fusion protein (e.g., a Cas9 domain fused to an thymine alkyltransferase domain) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair. As a result of embodiments of these methods, strand separation of said target region is induced, a first nucleobase of said target nucleobase pair in a single strand of the target region is converted to a second nucleobase, and no more than one strand of said target region is cut (or nicked), wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase..

[0332] In some embodiments, the first nucleobase is a thymine (of the target T: A nucleobase pair). In some embodiments, the second nucleobase is one of the intermediates N3-methyl thymine, N3-carboxymethyl thymine, or N3-3-amino-3-carboxypropyl thymine. In some embodiments, the third nucleobase is also an adenine (of the target T: A base pair). In some embodiments, the fourth nucleobase is a thymine (of the A:T pair). In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase (thymine) that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., T:A to an A:T pair). In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base paires are edited.

[0333] In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some

embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a editing window.

[0334] In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair (e.g., T:A target base pair), b) converting a first nucleobase (e.g., the T base) of said target nucleobase pair in a single strand of the target region to a second nucleobase (e.g., converted to an intermediate, such as, N3-methyl thymine, which is then replaced with an A through DNA replication/repair processes), c) cutting (or nicking) no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%.

[0335] In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. In other embodiments, the method results in less than 35% indel formation in the nucleic acid.

In some embodiments, at least 5% of the intended base pairs are edited. In some

embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase.

In some embodiments, the nucleobase editor comprises thymine alkylation, and/or DNA alkylation repair inhibition activity. In some embodiments, the nucleobase editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some

embodiments, the intended edited basepair is downstream of a PAM site. In some

embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some

embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1- 5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein.

[0336] In another embodiment, the disclosure provides editing methods comprising contacting a DNA, or RNA molecule with any of the base editors provided herein, and with at least one guide nucleic acid (e.g., guide RNA), wherein the guide nucleic acid, (e.g., guide RNA) is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

[0337] In some embodiments, the target nucleic acid sequence comprises a sequence associated with a disease, disorder, or condition. In some embodiments, the target nucleic acid sequence comprises a point mutation associated with a disease, disorder, or condition.

In some embodiments, the activity of the fusion protein (e.g., comprising an thymine alkyltransferase and a Cas9 domain), or the complex, results in a correction of the point mutation. In some embodiments, the target nucleic acid sequence comprises an A T point mutation associated with a disease, disorder, or condition, and wherein the conversion of the mutant T to an A results in a sequence that is not associated with a disease, disorder, or condition. In other embodiments, the target sequence comprises a T A point mutation associated with a disease, disorder, or condition, and wherein the conversion of the mutant A to a T results in a sequence that is not associated with a disease, disorder, or condition. In some embodiments, the target nucleic acid sequence encodes a protein, and the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon.

[0338] In some embodiments, the transversion of the mutant T (or mutant A) results in a change of the amino acid encoded by the mutant codon. In some embodiments, the transversion of the mutant T (or mutant A) results in the codon encoding the wild-type amino acid. In some embodiments, the step of contacting is performed in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease, disorder, or condition. In some embodiments, the disease, disorder, or condition is sickle cell anemia, Fanconi anemia, ectodermal dysplasia skin fragility syndrome, lattice comeal dystrophy Type III, or Noonan syndrome.

[0339] Some embodiments provide methods for using the base editors provided herein. In some embodiments, the base editors are used to introduce a point mutation into a nucleic acid by alkylating a target T nucleobase. In some embodiments, the alkylation of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease, disorder, or condition, e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes. In some embodiments, the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease, disorder, or condition. For example, in some embodiments, methods are provided herein that employ a DNA editing fusion protein to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease). A deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein.

[0340] In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional gene via genome editing. The base editor proteins provided herein may be validated for gene editing-based human therapeutics in vitro , e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the base editor proteins provided herein, e.g., the fusion proteins comprising a nucleic acid programmable DNA binding protein (e.g., Cas9) and a nucleobase modification domain may be used to correct any single point T to A mutation. Alkylation of a mutant T, followed by a round of replication, corrects the mutation.

[0341] The successful correction of point mutations in disease-associated genes and alleles opens up new strategies for gene correction with applications in therapeutics and basic research. Site-specific single-base modification systems like the disclosed fusions of a nucleic acid programmable DNA binding protein and an thymine alkyltransferase domain also have applications in“reverse” gene therapy, where certain gene functions are purposely suppressed or abolished. In these cases, site-specifically mutating residues that lead to inactivating mutations in a protein, or mutations that inhibit function of the protein may be used to abolish or inhibit protein function.

Methods of Treatment

[0342] The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a DNA editing fusion protein provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of an thymine alkyltransferase fusion protein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene. In some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of an thymine alkyltransferase fusion protein that corrects the point mutation or introduces a deactivating mutation into a disease- associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease- associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.

[0343] The instant disclosure provides methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by thymine alkyltransferase-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Exemplary suitable diseases and disorders include, without limitation: 2-methyl-3-hydroxybutyric aciduria; 3 beta- Hydroxysteroid dehydrogenase deficiency; 3-Methylglutaconic aciduria; 3-Oxo-5 alpha- steroid delta 4-dehydrogenase deficiency; 46, XY sex reversal, type 1, 3, and 5; 5- Oxoprolinase deficiency; 6-pyruvoyl-tetrahydropterin synthase deficiency; Aarskog syndrome; Aase syndrome; Achondrogenesis type 2; Achromatopsia 2 and 7; Acquired long QT syndrome; Acrocallosal syndrome, Schinzel type; Acrocapitofemoral dysplasia;

Acrodysostosis 2, with or without hormone resistance; Acroerythrokeratoderma; Acromicric dysplasia; Acth-independent macronodular adrenal hyperplasia 2; Activated PI3K-delta syndrome; Acute intermittent porphyria; deficiency of Acyl-CoA dehydrogenase family, member 9; Adams-Oliver syndrome 5 and 6; Adenine phosphoribosyltransferase deficiency; Adenylate kinase deficiency; hemolytic anemia due to Adenylosuccinate lyase deficiency; Adolescent nephronophthisis; Renal-hepatic-pancreatic dysplasia; Meckel syndrome type 7; Adrenoleukodystrophy; Adult junctional epidermolysis bullosa; Epidermolysis bullosa, junctional, localisata variant; Adult neuronal ceroid lipofuscinosis; Adult neuronal ceroid lipofuscinosis; Adult onset ataxia with oculomotor apraxia; ADULT syndrome; Afibrinogenemia and congenital Afibrinogenemia; autosomal recessive

Agammaglobulinemia 2; Age-related macular degeneration 3, 6, 11, and 12; Aicardi

Goutieres syndromes 1, 4, and 5; Chilbain lupus 1; Alagille syndromes 1 and 2; Alexander disease; Alkaptonuria; Allan-Herndon-Dudley syndrome; Alopecia universalis congenital; Alpers encephalopathy; Alpha- 1 -antitrypsin deficiency; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndromes; Alzheimer disease, familial, 3, with spastic paraparesis and apraxia; Alzheimer disease, types, 1, 3, and 4; hypocalcification type and hypomaturation type, IIA1 Amelogenesis imperfecta; Aminoacylase 1 deficiency; Amish infantile epilepsy syndrome; Amyloidogenic transthyretin amyloidosis; Amyloid

Cardiomyopathy, Transthyretin-related; Cardiomyopathy; Amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia), 22 (with or without frontotemporal dementia), and 10; Frontotemporal dementia with TDP43 inclusions, TARDBP-related; Andermann syndrome; Andersen Tawil syndrome; Congenital long QT syndrome; Anemia, nonspherocytic hemolytic, due to G6PD deficiency; Angelman syndrome; Severe neonatal- onset encephalopathy with microcephaly; susceptibility to Autism, X-linked 3; Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps; Angiotensin i-converting enzyme, benign serum increase; Aniridia, cerebellar ataxia, and mental retardation;

Anonychia; Antithrombin III deficiency; Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis; Aortic aneurysm, familial thoracic 4, 6, and 9; Thoracic aortic aneurysms and aortic dissections; Multisystemic smooth muscle dysfunction syndrome; Moyamoya disease 5; Aplastic anemia; Apparent mineralocorticoid excess; Arginase deficiency; Argininosuccinate lyase deficiency; Aromatase deficiency; Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10; Primary familial hypertrophic

cardiomyopathy; Arthrogryposis multiplex congenita, distal, X-linked; Arthrogryposis renal dysfunction cholestasis syndrome; Arthrogryposis, renal dysfunction, and cholestasis 2; Asparagine synthetase deficiency; Abnormality of neuronal migration; Ataxia with vitamin E deficiency; Ataxia, sensory, autosomal dominant; Ataxia-telangiectasia syndrome; Hereditary cancer-predisposing syndrome; Atransferrinemia; Atrial fibrillation, familial, 11, 12, 13, and 16; Atrial septal defects 2, 4, and 7 (with or without atrioventricular conduction defects); Atrial standstill 2; Atrioventricular septal defect 4; Atrophia bulborum hereditaria; ATR-X syndrome; Auriculocondylar syndrome 2; Autoimmune disease, multisystem, infantile-onset; Autoimmune lymphoproliferative syndrome, type la; Autosomal dominant hypohidrotic ectodermal dysplasia; Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions 1 and 3; Autosomal dominant torsion dystonia 4; Autosomal recessive centronuclear myopathy; Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; Autosomal recessive cutis laxa type IA and IB; Autosomal recessive hypohidrotic ectodermal dysplasia syndrome; Ectodermal dysplasia l ib; hypohidrotic/hair/tooth type, autosomal recessive; Autosomal recessive hypophosphatemic bone disease; Axenfeld-Rieger syndrome type 3; Bainbridge-Ropers syndrome; Bannayan-Riley-Ruvalcaba syndrome;

PTEN hamartoma tumor syndrome; Baraitser-Winter syndromes 1 and 2; Barakat syndrome; Bardet-Biedl syndromes 1, 11, 16, and 19; Bare lymphocyte syndrome type 2,

complementation group E; Bartter syndrome antenatal type 2; Bartter syndrome types 3, 3 with hypocalciuria , and 4; Basal ganglia calcification, idiopathic, 4; Beaded hair; Benign familial hematuria; Benign familial neonatal seizures 1 and 2; Seizures, benign familial neonatal, 1, and/or myokymia; Seizures, Early infantile epileptic encephalopathy 7; Benign familial neonatal-infantile seizures; Benign hereditary chorea; Benign scapuloperoneal muscular dystrophy with cardiomyopathy; Bemard-Soulier syndrome, types A1 and A2 (autosomal dominant); Bestrophinopathy, autosomal recessive; beta Thalassemia; Bethlem myopathy and Bethlem myopathy 2; Bietti crystalline corneoretinal dystrophy; Bile acid synthesis defect, congenital, 2; Biotinidase deficiency; Birk Barel mental retardation dysmorphism syndrome; Blepharophimosis, ptosis, and epicanthus inversus; Bloom syndrome; Borjeson-Forssman-Lehmann syndrome; Boucher Neuhauser syndrome;

Brachydactyly types A1 and A2; Brachydactyly with hypertension; Brain small vessel disease with hemorrhage; Branched-chain ketoacid dehydrogenase kinase deficiency;

Branchiootic syndromes 2 and 3; Breast cancer, early-onset; Breast-ovarian cancer, familial 1, 2, and 4; Brittle cornea syndrome 2; Brody myopathy; Bronchiectasis with or without elevated sweat chloride 3; Brown- Vialetto- Van laere syndrome and Brown- Vialetto- Van Laere syndrome 2; Brugada syndrome; Brugada syndrome 1; Ventricular fibrillation;

Paroxysmal familial ventricular fibrillation; Brugada syndrome and Brugada syndrome 4; Long QT syndrome; Sudden cardiac death; Bull eye macular dystrophy; Stargardt disease 4; Cone-rod dystrophy 12; Bullous ichthyosiform erythroderma; Burn-Mckeown syndrome; Candidiasis, familial, 2, 5, 6, and 8; Carbohydrate-deficient glycoprotein syndrome type I and II; Carbonic anhydrase VA deficiency, hyperammonemia due to; Carcinoma of colon;

Cardiac arrhythmia; Long QT syndrome, LQT1 subtype; Cardioencephalo myopathy, fatal infantile, due to cytochrome c oxidase deficiency; Cardiofaciocutaneous syndrome;

Cardiomyopathy; Danon disease; Hypertrophic cardiomyopathy; Left ventricular

noncompaction cardiomyopathy; Camevale syndrome; Carney complex, type 1; Carnitine acylcarnitine translocase deficiency; Carnitine palmitoyltransferase I , II, II (late onset), and II (infantile) deficiency; Cataract 1, 4, autosomal dominant, autosomal dominant, multiple types, with microcornea, coppock-like, juvenile, with microcornea and glucosuria, and nuclear diffuse nonprogressive; Catecholaminergic polymorphic ventricular tachycardia; Caudal regression syndrome; Cd8 deficiency, familial; Central core disease; Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency; Cerebellar ataxia infantile with progressive external ophthalmoplegi and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome 2; Cerebral amyloid angiopathy, APP-related; Cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations 2; Cerebrooculofacioskeletal syndrome 2; Cerebro-oculo- facio- skeletal syndrome; Cerebroretinal microangiopathy with calcifications and cysts;

Ceroid lipofuscinosis neuronal 2, 6, 7, and 10; Ch\xc3\xa9diak-Higashi syndrome , Chediak- Higashi syndrome, adult type; Charcot-Marie-Tooth disease types IB, 2B2, 2C, 2F, 21, 2U (axonal), 1C (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF, and X; Scapuloperoneal spinal muscular atrophy; Distal spinal muscular atrophy, congenital nonprogressive; Spinal muscular atrophy, distal, autosomal recessive, 5; CHARGE association; Childhood hypophosphatasia; Adult hypophosphatasia; Cholecystitis; Progressive familial intrahepatic cholestasis 3; Cholestasis, intrahepatic, of pregnancy 3; Cholestanol storage disease; Cholesterol monooxygenase (side-chain cleaving) deficiency; Chondrodysplasia Blomstrand type; Chondrodysplasia punctata 1, X-linked recessive and 2 X-linked dominant; CHOPS syndrome; Chronic granulomatous disease, autosomal recessive cytochrome b-positive, types 1 and 2; Chudley-McCullough syndrome; Ciliary dyskinesia, primary, 7, 11, 15, 20 and 22; Citrullinemia type I; Citrullinemia type I and II; Cleidocranial dysostosis; C-like syndrome; Cockayne syndrome type A, ; Coenzyme Q10 deficiency, primary 1, 4, and 7; Coffin Siris/Intellectual Disability; Coffin-Lowry syndrome; Cohen syndrome, ; Cold-induced sweating syndrome 1; COLE-CARPENTER SYNDROME 2; Combined cellular and humoral immune defects with granulomas; Combined d-2- and 1-2- hydroxyglutaric aciduria; Combined malonic and methylmalonic aciduria; Combined oxidative phosphorylation deficiencies 1, 3, 4, 12, 15, and 25; Combined partial and complete 17-alpha-hydroxylase/ 17, 20-lyase deficiency; Common variable immunodeficiency 9;

Complement component 4, partial deficiency of, due to dysfunctional cl inhibitor;

Complement factor B deficiency; Cone monochromatism; Cone-rod dystrophy 2 and 6;

Cone-rod dystrophy amelogenesis imperfecta; Congenital adrenal hyperplasia and Congenital adrenal hypoplasia, X-linked; Congenital amegakaryocytic thrombocytopenia; Congenital aniridia; Congenital central hypoventilation; Hirschsprung disease 3; Congenital contractural arachnodactyly; Congenital contractures of the limbs and face, hypotonia, and developmental delay; Congenital disorder of glycosylation types IB, ID, 1G, 1H, 1 J, IK, IN, IP, 2C, 2J,

2K, Urn; Congenital dyserythropoietic anemia, type I and II; Congenital ectodermal dysplasia of face; Congenital erythropoietic porphyria; Congenital generalized lipodystrophy type 2; Congenital heart disease, multiple types, 2; Congenital heart disease; Interrupted aortic arch; Congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; Non-small cell lung cancer; Neoplasm of ovary; Cardiac conduction defect, nonspecific; Congenital microvillous atrophy; Congenital muscular dystrophy; Congenital muscular dystrophy due to partial LAMA2 deficiency; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, types A2, A7, A8, Al l, and A14; Congenital muscular dystrophy- dystroglycanopathy with mental retardation, types B2, B3, B5, and B15; Congenital muscular dystrophy-dystroglycanopathy without mental retardation, type B5; Congenital muscular hypertrophy-cerebral syndrome; Congenital myasthenic syndrome, acetazolamide- responsive; Congenital myopathy with fiber type disproportion; Congenital ocular coloboma; Congenital stationary night blindness, type 1A, IB, 1C, IE, IF, and 2A; Coproporphyria; Cornea plana 2; Comeal dystrophy, Fuchs endothelial, 4; Comeal endothelial dystrophy type 2; Comeal fragility keratoglobus, blue sclerae and joint hypermobility; Cornelia de Fange syndromes 1 and 5; Coronary artery disease, autosomal dominant 2; Coronary heart disease; Hyperalphalipoproteinemia 2; Cortical dysplasia, complex, with other brain malformations 5 and 6; Cortical malformations, occipital; Corticosteroid-binding globulin deficiency;

Corticosterone methyloxidase type 2 deficiency; Costello syndrome; Cowden syndrome 1; Coxa plana; Craniodiaphyseal dysplasia, autosomal dominant; Craniosynostosis 1 and 4; Craniosynostosis and dental anomalies; Creatine deficiency, X-linked; Crouzon syndrome; Cryptophthalmos syndrome; Cryptorchidism, unilateral or bilateral; Cushing symphalangism; Cutaneous malignant melanoma 1; Cutis laxa with osteodystrophy and with severe pulmonary, gastrointestinal, and urinary abnormalities; Cyanosis, transient neonatal and atypical nephropathic; Cystic fibrosis; Cystinuria; Cytochrome c oxidase i deficiency;

Cytochrome-c oxidase deficiency ; D-2-hydroxyglutaric aciduria 2; Darier disease, segmental; Deafness with labyrinthine aplasia microtia and microdontia (FAMM); Deafness, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17, 20, and 65; Deafness, autosomal recessive 1A, 2, 3, 6, 8, 9, 12, 15, 16, 18b, 22, 28, 31, 44,

49, 63, 77, 86, and 89; Deafness, cochlear, with myopia and intellectual impairment, without vestibular involvement, autosomal dominant, X-linked 2; Deficiency of 2-methylbutyryl-CoA dehydrogenase; Deficiency of 3-hydroxyacyl-CoA dehydrogenase; Deficiency of alpha- mannosidase; Deficiency of aromatic-L-amino-acid decarboxylase; Deficiency of

bisphosphoglycerate mutase; Deficiency of butyryl-CoA dehydrogenase; Deficiency of ferroxidase; Deficiency of galactokinase; Deficiency of guanidinoacetate methyltransferase; Deficiency of hyaluronoglucosaminidase; Deficiency of ribose-5-phosphate isomerase;

Deficiency of steroid 11 -beta-monooxygenase; Deficiency of UDPglucose-hexose-1- phosphate uridylyltransferase; Deficiency of xanthine oxidase; Dejerine-Sottas disease;

Charcot-Marie-Tooth disease, types ID and IVF; Dejerine-Sottas syndrome, autosomal dominant; Dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiency; Desbuquois dysplasia 2; Desbuquois syndrome; DFNA 2 Nonsyndromic Hearing Loss;

Diabetes mellitus and insipidus with optic atrophy and deafness; Diabetes mellitus, type 2, and insulin-dependent, 20; Diamond-Blackfan anemia 1, 5, 8, and 10; Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tufting enteropathy, congenital); Dicarboxylic aminoaciduria; Diffuse palmoplantar keratoderma, Bothnian type; Digitorenocerebral syndrome; Dihydropteridine reductase deficiency; Dilated cardiomyopathy 1A, 1AA, 1C, 1G, IBB, 1DD, IFF, 1HH, II, IKK, IN, IS, 1Y, and 3B; Left ventricular noncompaction 3; Disordered steroidogenesis due to cytochrome p450 oxidoreductase deficiency; Distal arthrogryposis type 2B; Distal hereditary motor neuronopathy type 2B; Distal myopathy Markesbery-Griggs type; Distal spinal muscular atrophy, X-linked 3; Distichiasis- lymphedema syndrome; Dominant dystrophic epidermolysis bullosa with absence of skin; Dominant hereditary optic atrophy; Donnai Barrow syndrome; Dopamine beta hydroxylase deficiency; Dopamine receptor d2, reduced brain density of; Dowling-degos disease 4; Doyne honeycomb retinal dystrophy; Malattia leventinese; Duane syndrome type 2; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Becker muscular dystrophy; Dysfibrinogenemia; Dyskeratosis congenita autosomal dominant and autosomal dominant, 3; Dyskeratosis congenita, autosomal recessive, 1, 3, 4, and 5; Dyskeratosis congenita X-linked; Dyskinesia, familial, with facial myokymia; Dysplasminogenemia; Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (Dopa-responsive type ), 10, 12, 16, 25, 26 (Myoclonic); Seizures, benign familial infantile, 2; Early infantile epileptic encephalopathy 2, 4, 7, 9, 10,

11, 13, and 14; Atypical Rett syndrome; Early T cell progenitor acute lymphoblastic leukemia; Ectodermal dysplasia skin fragility syndrome; Ectodermal dysplasia-syndactyly syndrome 1; Ectopia lentis, isolated autosomal recessive and dominant; Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3; Ehlers-Danlos syndrome type 7 (autosomal recessive), classic type, type 2 (progeroid ), hydroxylysine-deficient, type 4, type 4 variant, and due to tenascin-X deficiency; Eichsfeld type congenital muscular dystrophy; Endocrine-cerebroosteodysplasia; Enhanced s-cone syndrome; Enlarged vestibular aqueduct syndrome; Enterokinase deficiency; Epidermodysplasia verruciformis; Epidermolysa bullosa simplex and limb girdle muscular dystrophy, simplex with mottled pigmentation, simplex with pyloric atresia, simplex, autosomal recessive, and with pyloric atresia; Epidermolytic palmoplantar keratoderma; Familial febrile seizures 8; Epilepsy, childhood absence 2, 12 (idiopathic generalized, susceptibility to) 5 (nocturnal frontal lobe), nocturnal frontal lobe type 1, partial, with variable foci, progressive myoclonic 3, and X-linked, with variable learning disabilities and behavior disorders; Epileptic encephalopathy, childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Epiphyseal dysplasia, multiple, with myopia and conductive deafness; Episodic ataxia type 2; Episodic pain syndrome, familial, 3; Epstein syndrome; Fechtner syndrome; Erythropoietic protoporphyria; Estrogen resistance; Exudative vitreoretinopathy 6; Fabry disease and Fabry disease, cardiac variant; Factor H, VII, X, v and factor viii, combined deficiency of 2, xiii, a subunit, deficiency; Familial adenomatous polyposis 1 and 3; Familial amyloid nephropathy with urticaria and deafness; Familial cold urticarial; Familial aplasia of the vermis; Familial benign pemphigus; Familial cancer of breast; Breast cancer, susceptibility to; Osteosarcoma; Pancreatic cancer 3; Familial cardiomyopathy; Familial cold autoinflammatory syndrome 2; Familial colorectal cancer; Familial exudative vitreoretinopathy, X-linked; Familial hemiplegic migraine types 1 and 2; Familial hypercholesterolemia; Familial hypertrophic cardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24; Familial hypokalemia-hypomagnesemia; Familial hypoplastic, glomerulocystic kidney; Familial infantile myasthenia; Familial juvenile gout; Familial Mediterranean fever and Familial mediterranean fever, autosomal dominant; Familial porencephaly; Familial porphyria cutanea tarda; Familial pulmonary capillary hemangiomatosis; Familial renal glucosuria; Familial renal hypouricemia; Familial restrictive cardiomyopathy 1; Familial type 1 and 3 hyperlipoproteinemia; Fanconi anemia, complementation group E, I, N, and O; Fanconi-Bickel syndrome; Favism, susceptibility to; Febrile seizures, familial, 11; Feingold syndrome 1; Fetal hemoglobin quantitative trait locus 1; FG syndrome and FG syndrome 4; Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with or without extraocular

involvement), 3b; Fish-eye disease; Fleck comeal dystrophy; Floating-Harbor syndrome; Focal epilepsy with speech disorder with or without mental retardation; Focal segmental glomerulosclerosis 5; Forebrain defects; Frank Ter Haar syndrome; Borrone Di Rocco Crovato syndrome; Frasier syndrome; Wilms tumor 1; Freeman-Sheldon syndrome;

Frontometaphyseal dysplasia land 3; Frontotemporal dementia; Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4; Frontotemporal Dementia Chromosome 3- Linked and Frontotemporal dementia ubiquitin-positive; Fructose-biphosphatase deficiency; Fuhrmann syndrome; Gamma-aminobutyric acid transaminase deficiency; Gamstorp- Wohlfart syndrome; Gaucher disease type 1 and Subacute neuronopathic; Gaze palsy, familial horizontal, with progressive scoliosis; Generalized dominant dystrophic

epidermolysis bullosa; Generalized epilepsy with febrile seizures plus 3, type 1, type 2; Epileptic encephalopathy Lennox-Gastaut type; Giant axonal neuropathy; Glanzmann thrombasthenia; Glaucoma 1, open angle, e, F, and G; Glaucoma 3, primary congenital, d; Glaucoma, congenital and Glaucoma, congenital, Coloboma; Glaucoma, primary open angle, juvenile-onset; Glioma susceptibility 1; Glucose transporter type 1 deficiency syndrome; Glucose-6-phosphate transport defect; GLUT1 deficiency syndrome 2; Epilepsy, idiopathic generalized, susceptibility to, 12; Glutamate formiminotransferase deficiency; Glutaric acidemia IIA and IIB; Glutaric aciduria, type 1; Gluthathione synthetase deficiency;

Glycogen storage disease 0 ( muscle), II (adult form), IXa2, IXc, type 1A; type II, type IV,

IV (combined hepatic and myopathic), type V, and type VI; Goldmann-Favre syndrome; Gordon syndrome; Gorlin syndrome; Holoprosencephaly sequence; Holoprosencephaly 7; Granulomatous disease, chronic, X-linked, variant; Granulosa cell tumor of the ovary; Gray platelet syndrome; Griscelli syndrome type 3; Groenouw comeal dystrophy type I; Growth and mental retardation, mandibulofacial dysostosis, microcephaly, and cleft palate; Growth hormone deficiency with pituitary anomalies; Growth hormone insensitivity with

immunodeficiency; GTP cyclohydrolase I deficiency; Hajdu-Cheney syndrome; Hand foot uterus syndrome; Hearing impairment; Hemangioma, capillary infantile; Hematologic neoplasm; Hemochromatosis type 1, 2B, and 3; Microvascular complications of diabetes 7; Transferrin serum level quantitative trait locus 2; Hemoglobin H disease, nondeletional; Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency;

Hemophagocytic lymphohistiocytosis, familial, 2; Hemophagocytic lymphohistiocytosis, familial, 3; Heparin cofactor II deficiency; Hereditary acrodermatitis enteropathica;

Hereditary breast and ovarian cancer syndrome; Ataxia-telangiectasia-like disorder;

Hereditary diffuse gastric cancer; Hereditary diffuse leukoencephalopathy with spheroids; Hereditary factors II, IX, VIII deficiency disease; Hereditary hemorrhagic telangiectasia type 2; Hereditary insensitivity to pain with anhidrosis; Hereditary lymphedema type I; Hereditary motor and sensory neuropathy with optic atrophy; Hereditary myopathy with early respiratory failure; Hereditary neuralgic amyotrophy; Hereditary Nonpolyposis Colorectal Neoplasms; Lynch syndrome I and II; Hereditary pancreatitis; Pancreatitis, chronic, susceptibility to; Hereditary sensory and autonomic neuropathy type IIB amd IIA; Hereditary sideroblastic anemia; Hermansky-Pudlak syndrome 1, 3, 4, and 6; Heterotaxy, visceral, 2, 4, and 6, autosomal; Heterotaxy, visceral, X-linked; Heterotopia; Histiocytic medullary reticulosis; Histiocytosis-lymphadenopathy plus syndrome; Holocarboxylase synthetase deficiency; Holoprosencephaly 2, 3,7, and 9; Holt-Oram syndrome; Homocysteinemia due to MTHFR deficiency, CBS deficiency, and Homocystinuria, pyridoxine-responsive;

Homocystinuria-Megaloblastic anemia due to defect in cobalamin metabolism, cblE complementation type; Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome; Hydrocephalus; Hyperammonemia, type III; Hypercholesterolaemia and

Hypercholesterolemia, autosomal recessive; Hyperekplexia 2 and Hyperekplexia hereditary; Hyperferritinemia cataract syndrome; Hyperglycinuria; Hyperimmunoglobulin D with periodic fever; Mevalonic aciduria; Hyperimmunoglobulin E syndrome; Hyperinsulinemic hypoglycemia familial 3, 4, and 5; Hyperinsulinism-hyperammonemia syndrome;

Hyperlysinemia; Hypermanganesemia with dystonia, polycythemia and cirrhosis;

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; Hyperparathyroidism 1 and 2; Hyperparathyroidism, neonatal severe; Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency, BH4-deficient, D, and non-pku; Hyperphosphatasia with mental retardation syndrome 2, 3, and 4; Hypertrichotic osteochondrodysplasia;

Hypobetalipoproteinemia, familial, associated with apob32; Hypocalcemia, autosomal dominant 1; Hypocalciuric hypercalcemia, familial, types 1 and 3; Hypochondrogenesis; Hypochromic microcytic anemia with iron overload; Hypoglycemia with deficiency of glycogen synthetase in the liver; Hypogonadotropic hypogonadism 11 with or without anosmia; Hypohidrotic ectodermal dysplasia with immune deficiency; Hypohidrotic X-linked ectodermal dysplasia; Hypokalemic periodic paralysis 1 and 2; Hypomagnesemia 1, intestinal; Hypomagnesemia, seizures, and mental retardation; Hypomyelinating

leukodystrophy 7; Hypoplastic left heart syndrome; Atrioventricular septal defect and common atrioventricular junction; Hypospadias 1 and 2, X-linked; Hypothyroidism, congenital, nongoitrous, 1; Hypotrichosis 8 and 12; Hypotrichosis-lymphedema- telangiectasia syndrome; I blood group system; Ichthyosis bullosa of Siemens; Ichthyosis exfoliativa; Ichthyosis prematurity syndrome; Idiopathic basal ganglia calcification 5;

Idiopathic fibrosing alveolitis, chronic form; Dyskeratosis congenita, autosomal dominant, 2 and 5; Idiopathic hypercalcemia of infancy; Immune dysfunction with T-cell inactivation due to calcium entry defect 2; Immunodeficiency 15, 16, 19, 30, 31C, 38, 40, 8, due to defect in cd3-zeta, with hyper IgM type 1 and 2, and X-Linked, with magnesium defect, Epstein-Barr virus infection, and neoplasia; Immunodeficiency-centromeric instability-facial anomalies syndrome 2; Inclusion body myopathy 2 and 3; Nonaka myopathy; Infantile convulsions and paroxysmal choreoathetosis, familial; Infantile cortical hyperostosis; Infantile GM1 gangliosidosis; Infantile hypophosphatasia; Infantile nephronophthisis; Infantile nystagmus, X-linked; Infantile Parkinsonism-dystonia; Infertility associated with multi-tailed

spermatozoa and excessive DNA; Insulin resistance; Insulin-resistant diabetes mellitus and acanthosis nigricans; Insulin-dependent diabetes mellitus secretory diarrhea syndrome;

Interstitial nephritis, karyomegalic; Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies; Iodotyrosyl coupling defect; IRAK4 deficiency; Iridogoniodysgenesis dominant type and type 1; Iron accumulation in brain; Ischiopatellar dysplasia; Islet cell hyperplasia; Isolated 17,20-lyase deficiency; Isolated lutropin deficiency; Isovaleryl-CoA dehydrogenase deficiency; Jankovic Rivera syndrome; Jervell and Lange-Nielsen syndrome 2; Joubert syndrome 1, 6, 7, 9/15 (digenic), 14, 16, and 17, and Orofaciodigital syndrome xiv; Junctional epidermolysis bullosa gravis of Herlitz; Juvenile GM>1< gangliosidosis; Juvenile polyposis syndrome; Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome; Juvenile retinoschisis; Kabuki make-up syndrome; Kallmann syndrome 1, 2, and 6; Delayed puberty; Kanzaki disease; Karak syndrome;

Kartagener syndrome; Kenny-Caffey syndrome type 2; Keppen-Lubinsky syndrome;

Keratoconus 1; Keratosis follicularis; Keratosis palmoplantaris striata 1; Kindler syndrome; L-2-hydroxyglutaric aciduria; Larsen syndrome, dominant type; Lattice corneal dystrophy Type III; Leber amaurosis; Zellweger syndrome; Peroxisome biogenesis disorders; Zellweger syndrome spectrum; Leber congenital amaurosis 11, 12, 13, 16, 4, 7, and 9; Leber optic atrophy; Aminoglycoside-induced deafness; Deafness, nonsyndromic sensorineural, mitochondrial; Left ventricular noncompaction 5; Left-right axis malformations; Leigh disease; Mitochondrial short-chain Enoyl-CoA Hydratase 1 deficiency; Leigh syndrome due to mitochondrial complex I deficiency; Leiner disease; Leri Weill dyschondrosteosis; Lethal congenital contracture syndrome 6; Leukocyte adhesion deficiency type I and III;

Leukodystrophy, Hypomyelinating, 11 and 6; Leukoencephalopathy with ataxia, with Brainstem and Spinal Cord Involvement and Lactate Elevation, with vanishing white matter, and progressive, with ovarian failure; Leukonychia totalis; Lewy body dementia;

Lichtenstein-Knorr Syndrome; Li-Fraumeni syndrome 1; Lig4 syndrome; Limb-girdle muscular dystrophy, type IB, 2A, 2B, 2D, Cl, C5, C9, C14; Congenital muscular dystrophy- dystroglycanopathy with brain and eye anomalies, type A14 and B14; Lipase deficiency combined; Lipid proteinosis; Lipodystrophy, familial partial, type 2 and 3; Lissencephaly 1, 2 (X-linked), 3, 6 (with microcephaly), X-linked; Subcortical laminar heterotopia, X-linked; Liver failure acute infantile; Loeys-Dietz syndrome 1, 2, 3; Long QT syndrome 1, 2, 2/9, 2/5, (digenic), 3, 5 and 5, acquired, susceptibility to; Lung cancer; Lymphedema, hereditary, id; Lymphedema, primary, with myelodysplasia; Lymphoproliferative syndrome 1, 1 (X-linked), and 2; Lysosomal acid lipase deficiency; Macrocephaly, macrosomia, facial dysmorphism syndrome; Macular dystrophy, vitelliform, adult-onset; Malignant hyperthermia susceptibility type 1; Malignant lymphoma, non-Hodgkin; Malignant melanoma; Malignant tumor of prostate; Mandibuloacral dysostosis; Mandibuloacral dysplasia with type A or B

lipodystrophy, atypical; Mandibulofacial dysostosis, Treacher Collins type, autosomal recessive; Mannose-binding protein deficiency; Maple syrup urine disease type 1A and type 3; Marden Walker like syndrome; Marfan syndrome; Marinesco-Sj\xc3\xb6gren syndrome; Martsolf syndrome; Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3, and type 9; May-Hegglin anomaly; MYH9 related disorders; Sebastian syndrome; McCune- Albright syndrome; Somatotroph adenoma; Sex cord-stromal tumor; Cushing syndrome; McKusick Kaufman syndrome; McLeod neuroacanthocytosis syndrome; Meckel-Gruber syndrome; Medium-chain acyl-coenzyme A dehydrogenase deficiency; Medulloblastoma; Megalencephalic leukoencephalopathy with subcortical cysts land 2a; Megalencephaly cutis marmorata telangiectatica congenital; PIK3CA Related Overgrowth Spectrum;

Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2; Megaloblastic anemia, thiamine-responsive, with diabetes mellitus and sensorineural deafness; Meier-Gorlin syndromes land 4; Melnick-Needles syndrome; Meningioma; Mental retardation, X-linked, 3, 21, 30, and 72; Mental retardation and microcephaly with pontine and cerebellar hypoplasia; Mental retardation X-linked syndromic 5; Mental retardation, anterior maxillary protrusion, and strabismus; Mental retardation, autosomal dominant 12, 13, 15, 24, 3, 30, 4,

5, 6, and 9; Mental retardation, autosomal recessive 15, 44, 46, and 5; Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations; Mental retardation, syndromic, Claes-Jensen type, X-linked; Mental retardation, X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type; Merosin deficient congenital muscular dystrophy; Metachromatic leukodystrophy juvenile, late infantile, and adult types;

Metachromatic leukodystrophy; Metatrophic dysplasia; Methemoglobinemia types I and 2; Methionine adenosyltransferase deficiency, autosomal dominant; Methylmalonic acidemia with homocystinuria, ; Methylmalonic aciduria cblB type, ; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency; METHYLMALONIC ACIDURIA, mut(0) TYPE; Microcephalic osteodysplastic primordial dwarfism type 2; Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation; Microcephaly, hiatal hernia and nephrotic syndrome; Microcephaly; Hypoplasia of the corpus callosum; Spastic paraplegia 50, autosomal recessive; Global developmental delay; CNS hypomyelination; Brain atrophy; Microcephaly, normal intelligence and immunodeficiency; Microcephaly-capillary malformation syndrome; Microcytic anemia; Microphthalmia syndromic 5, 7, and 9;

Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6; Microspherophakia; Migraine, familial basilar; Miller syndrome; Minicore myopathy with external ophthalmoplegia;

Myopathy, congenital with cores; Mitchell-Riley syndrome; mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase deficiency; Mitochondrial complex I, II, III, III (nuclear type 2, 4, or 8) deficiency; Mitochondrial DNA depletion syndrome 11, 12 (cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type); Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and 13 (encephalomyopathic type); Mitochondrial phosphate carrier and pyruvate carrier deficiency; Mitochondrial trifunctional protein deficiency; Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Miyoshi muscular dystrophy 1; Myopathy, distal, with anterior tibial onset; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency, complementation group A; Mowat-Wilson syndrome; Mucolipidosis III Gamma; Mucopolysaccharidosis type VI, type VI (severe), and type VII; Mucopolysaccharidosis, MPS-I-H/S, MPS-II, MPS-III-A, MPS-III-B, MPS-III-C, MPS-IV-A, MPS-IV-B; Retinitis Pigmentosa 73; Gangliosidosis GM1 typel (with cardiac involvenment) 3; Multicentric osteolysis nephropathy; Multicentric osteolysis, nodulosis and arthropathy; Multiple congenital anomalies; Atrial septal defect 2; Multiple congenital anomalies -hypotonia- seizures syndrome 3; Multiple Cutaneous and Mucosal Venous Malformations; Multiple endocrine neoplasia, types land 4; Multiple epiphyseal dysplasia 5 or Dominant; Multiple gastrointestinal atresias; Multiple pterygium syndrome Escobar type; Multiple sulfatase deficiency; Multiple synostoses syndrome 3; Muscle AMP thymine alkyltransferase deficiency; Muscle eye brain disease; Muscular dystrophy, congenital, megaconial type; Myasthenia, familial infantile, 1; Myasthenic Syndrome, Congenital, 11, associated with acetylcholine receptor deficiency; Myasthenic Syndrome, Congenital, 17, 2A (slow-channel), 4B (fast-channel), and without tubular aggregates; Myeloperoxidase deficiency; MYH- associated polyposis; Endometrial carcinoma; Myocardial infarction 1; Myoclonic dystonia; Myoclonic-Atonic Epilepsy; Myoclonus with epilepsy with ragged red fibers; Myofibrillar myopathy 1 and ZASP-related; Myoglobinuria, acute recurrent, autosomal recessive;

Myoneural gastrointestinal encephalopathy syndrome; Cerebellar ataxia infantile with progressive external ophthalmoplegia; Mitochondrial DNA depletion syndrome 4B, MNGIE type; Myopathy, centronuclear, 1, congenital, with excess of muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1, mitochondrial progressive with congenital cataract, hearing loss, and developmental delay, and tubular aggregate, 2; Myopia 6; Myosclerosis, autosomal recessive; Myotonia congenital; Congenital myotonia, autosomal dominant and recessive forms; Nail-patella syndrome; Nance-Horan syndrome; Nanophthalmos 2; Navajo neurohepatopathy; Nemaline myopathy 3 and 9; Neonatal hypotonia; Intellectual disability; Seizures; Delayed speech and language development; Mental retardation, autosomal dominant 31; Neonatal intrahepatic cholestasis caused by citrin deficiency; Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus, X-linked; Nephrolithiasis/osteoporosis, hypophosphatemic, 2; Nephronophthisis 13, 15 and 4; Infertility; Cerebello-oculo-renal syndrome (nephronophthisis, oculomotor apraxia and cerebellar abnormalities); Nephrotic syndrome, type 3, type 5, with or without ocular abnormalities, type 7, and type 9; Nestor- Guillermo progeria syndrome; Neu-Laxova syndrome 1; Neurodegeneration with brain iron accumulation 4 and 6; Neuroferritinopathy; Neurofibromatosis, type land type 2;

Neurofibrosarcoma; Neurohypophyseal diabetes insipidus; Neuropathy, Hereditary Sensory, Type IC; Neutral 1 amino acid transport defect; Neutral lipid storage disease with myopathy; Neutrophil immunodeficiency syndrome; Nicolaides-Baraitser syndrome; Niemann-Pick disease type Cl, C2, type A, and type Cl, adult form; Non-ketotic hyperglycinemia; Noonan syndrome 1 and 4, LEOPARD syndrome 1; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia; Normokalemic periodic paralysis, potassium-sensitive; Norum disease; Epilepsy, Hearing Loss, And Mental Retardation Syndrome; Mental Retardation, X-Linked 102 and syndromic 13; Obesity; Ocular albinism, type I;

Oculocutaneous albinism type IB, type 3, and type 4; Oculodentodigital dysplasia;

Odontohypophosphatasia; Odontotrichomelic syndrome; Oguchi disease; Oligodontia- colorectal cancer syndrome; Opitz G/BBB syndrome; Optic atrophy 9; Oral-facial-digital syndrome; Ornithine aminotransferase deficiency; Orofacial cleft 11 and 7, Cleft lip/palate- ectodermal dysplasia syndrome; Orstavik Lindemann Solberg syndrome; Osteoarthritis with mild chondrodysplasia; Osteochondritis dissecans; Osteogenesis imperfecta type 12, type 5, type 7, type 8, type I, type III, with normal sclerae, dominant form, recessive perinatal lethal; Osteopathia striata with cranial sclerosis; Osteopetrosis autosomal dominant type 1 and 2, recessive 4, recessive 1, recessive 6; Osteoporosis with pseudoglioma; Oto-palato-digital syndrome, types I and II; Ovarian dysgenesis 1; Ovarioleukodystrophy; Pachyonychia congenita 4 and type 2; Paget disease of bone, familial; Pallister-Hall syndrome;

Palmoplantar keratoderma, nonepidermolytic, focal or diffuse; Pancreatic agenesis and congenital heart disease; Papillon-Lef\xc3\xa8vre syndrome; Paragangliomas 3;

Paramyotonia congenita of von Eulenburg; Parathyroid carcinoma; Parkinson disease 14, 15, 19 (juvenile-onset), 2, 20 (early-onset), 6, (autosomal recessive early-onset, and 9; Partial albinism; Partial hypoxanthine-guanine phosphoribosyltransferase deficiency; Patterned dystrophy of retinal pigment epithelium; PC-K6a; Pelizaeus-Merzbacher disease; Pendred syndrome; Peripheral demyelinating neuropathy, central dysmyelination; Hirschsprung disease; Permanent neonatal diabetes mellitus; Diabetes mellitus, permanent neonatal, with neurologic features; Neonatal insulin-dependent diabetes mellitus; Maturity-onset diabetes of the young, type 2; Peroxisome biogenesis disorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B;

Perrault syndrome 4; Perry syndrome; Persistent hyperinsulinemic hypoglycemia of infancy; familial hyperinsulinism; Phenotypes; Phenylketonuria; Pheochromocytoma; Hereditary Paraganglioma- Pheochromocytoma Syndromes; Paragangliomas 1; Carcinoid tumor of intestine; Cowden syndrome 3; Phosphoglycerate dehydrogenase deficiency;

Phosphoglycerate kinase 1 deficiency; Photosensitive trichothiodystrophy; Phytanic acid storage disease; Pick disease; Pierson syndrome; Pigmentary retinal dystrophy; Pigmented nodular adrenocortical disease, primary, 1; Pilomatrixoma; Pitt- Hopkins syndrome; Pituitary dependent hypercortisolism; Pituitary hormone deficiency, combined 1, 2, 3, and 4;

Plasminogen activator inhibitor type 1 deficiency; Plasminogen deficiency, type I; Platelet- type bleeding disorder 15 and 8; Poikiloderma, hereditary fibrosing, with tendon contractures, myopathy, and pulmonary fibrosis; Polycystic kidney disease 2, adult type, and infantile type; Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy;

Polyglucosan body myopathy 1 with or without immunodeficiency; Polymicrogyria, asymmetric, bilateral frontoparietal; Polyneuropathy, hearing loss, ataxia, retinitis

pigmentosa, and cataract; Pontocerebellar hypoplasia type 4; Popliteal pterygium syndrome; Porencephaly 2; Porokeratosis 8, disseminated superficial actinic type; Porphobilinogen synthase deficiency; Porphyria cutanea tarda; Posterior column ataxia with retinitis pigmentosa; Posterior polar cataract type 2; Prader-Willi-like syndrome; Premature ovarian failure 4, 5, 7, and 9; Primary autosomal recessive microcephaly 10, 2, 3, and 5; Primary ciliary dyskinesia 24; Primary dilated cardiomyopathy; Left ventricular noncompaction 6; 4, Left ventricular noncompaction 10; Paroxysmal atrial fibrillation; Primary hyperoxaluria, type I, type, and type III; Primary hypertrophic osteoarthropathy, autosomal recessive 2; Primary hypomagnesemia; Primary open angle glaucoma juvenile onset 1; Primary pulmonary hypertension; Primrose syndrome; Progressive familial heart block type IB;

Progressive familial intrahepatic cholestasis 2 and 3; Progressive intrahepatic cholestasis; Progressive myoclonus epilepsy with ataxia; Progressive pseudorheumatoid dysplasia;

Progressive sclerosing poliodystrophy; Prolidase deficiency; Proline dehydrogenase deficiency; Schizophrenia 4; Properdin deficiency, X-linked; Propionic academia; Proprotein convertase 1/3 deficiency; Prostate cancer, hereditary, 2; Protan defect; Proteinuria; Finnish congenital nephrotic syndrome; Proteus syndrome; Breast adenocarcinoma;

Pseudoachondroplastic spondyloepiphyseal dysplasia syndrome; Pseudohypoaldosteronism type 1 autosomal dominant and recessive and type 2; Pseudohypoparathyroidism type 1A, Pseudopseudohypoparathyroidism; Pseudoneonatal adrenoleukodystrophy; Pseudoprimary hyperaldosteronism; Pseudoxanthoma elasticum; Generalized arterial calcification of infancy 2; Pseudoxanthoma elasticum-like disorder with multiple coagulation factor deficiency;

Psoriasis susceptibility 2; PTEN hamartoma tumor syndrome; Pulmonary arterial

hypertension related to hereditary hemorrhagic telangiectasia; Pulmonary Fibrosis And/Or Bone Marrow Failure, Telomere-Related, 1 and 3; Pulmonary hypertension, primary, 1, with hereditary hemorrhagic telangiectasia; Purine-nucleoside phosphorylase deficiency; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase El -alpha deficiency; Pyruvate kinase deficiency of red cells; Raine syndrome; Rasopathy; Recessive dystrophic epidermolysis bullosa; Nail disorder, nonsyndromic congenital, 8; Reifenstein syndrome; Renal adysplasia; Renal carnitine transport defect; Renal coloboma syndrome; Renal dysplasia; Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia; Renal tubular acidosis, distal, autosomal recessive, with late-onset sensorineural hearing loss, or with hemolytic anemia; Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; Retinal cone dystrophy 3B; Retinitis pigmentosa; Retinitis pigmentosa 10, 11, 12, 14, 15, 17, and 19; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48, 66, 7, 70, 72; Retinoblastoma; Rett disorder; Rhabdoid tumor predisposition syndrome 2;

Rhegmatogenous retinal detachment, autosomal dominant; Rhizomelic chondrodysplasia punctata type 2 and type 3; Roberts-SC phocomelia syndrome; Robinow Sorauf syndrome; Robinow syndrome, autosomal recessive, autosomal recessive, with brachy-syn-polydactyly; Rothmund-Thomson syndrome; Rapadilino syndrome; RRM2B-related mitochondrial disease; Rubinstein-Taybi syndrome; Salla disease; Sandhoff disease, adult and infantil types; Sarcoidosis, early-onset; Blau syndrome; Schindler disease, type 1; Schizencephaly;

Schizophrenia 15; Schneckenbecken dysplasia; Schwannomatosis 2; Schwartz Jampel syndrome type 1; Sclerocornea, autosomal recessive; Sclerosteosis; Secondary

hypothyroidism; Segawa syndrome, autosomal recessive; Senior-Loken syndrome 4 and 5, ; Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis; Sepiapterin reductase deficiency; SeSAME syndrome; Severe combined immunodeficiency due to ADA

deficiency, with microcephaly, growth retardation, and sensitivity to ionizing radiation, atypical, autosomal recessive, T cell-negative, B cell-positive, NK cell-negative of NK- positive; Severe congenital neutropenia; Severe congenital neutropenia 3, autosomal recessive or dominant; Severe congenital neutropenia and 6, autosomal recessive; Severe myoclonic epilepsy in infancy; Generalized epilepsy with febrile seizures plus, types 1 and 2; Severe X-linked myotubular myopathy; Short QT syndrome 3; Short stature with nonspecific skeletal abnormalities; Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities; Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis;

Primordial dwarfism; Short-rib thoracic dysplasia 11 or 3 with or without polydactyly;

Sialidosis type I and II; Sickle cell anemia; Silver spastic paraplegia syndrome; Slowed nerve conduction velocity, autosomal dominant; Smith-Lemli-Opitz syndrome; Snyder Robinson syndrome; Somatotroph adenoma; Prolactinoma; familial, Pituitary adenoma predisposition; Sotos syndrome 1 or 2; Spastic ataxia 5, autosomal recessive, Charlevoix-Saguenay type, 1,10, or 11, autosomal recessive; Amyotrophic lateral sclerosis type 5; Spastic paraplegia 15, 2, 3, 35, 39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Bile acid synthesis defect, congenital, 3; Spermatogenic failure 11, 3, and 8; Spherocytosis types 4 and 5;

Spheroid body myopathy; Spinal muscular atrophy, lower extremity predominant 2, autosomal dominant; Spinal muscular atrophy, type II; Spinocerebellar ataxia 14, 21, 35,

40, and 6; Spinocerebellar ataxia autosomal recessive 1 and 16; Splenic hypoplasia;

Spondylocarpotarsal synostosis syndrome; Spondylocheirodysplasia, Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type, with congenital joint

dislocations, short limb-hand type, Sedaghatian type, with cone-rod dystrophy, and

Kozlowski type; Parastremmatic dwarfism; Stargardt disease 1; Cone-rod dystrophy 3;

Stickler syndrome type 1; Kniest dysplasia; Stickler syndrome, types l(nonsyndromic ocular) and 4; Sting-associated vasculopathy, infantile-onset; Stormorken syndrome; Sturge -Weber syndrome, Capillary malformations, congenital, 1 ; Succinyl-CoA acetoacetate transferase deficiency; Sucrase-isomaltase deficiency; Sudden infant death syndrome; Sulfite oxidase deficiency, isolated; Supravalvar aortic stenosis; Surfactant metabolism dysfunction, pulmonary, 2 and 3; Symphalangism, proximal, lb; Syndactyly Cenani Lenz type;

Syndactyly type 3; Syndromic X-linked mental retardation 16; Talipes equinovarus; Tangier disease; TARP syndrome; Tay-Sachs disease, B1 variant, Gm2-gangliosidosis (adult), Gm2- gangliosidosis (adult-onset); Temtamy syndrome; Tenorio Syndrome; Terminal osseous dysplasia; Testosterone 17-beta-dehydrogenase deficiency; Tetraamelia, autosomal recessive; Tetralogy of Fallot; Hypoplastic left heart syndrome 2; Truncus arteriosus; Malformation of the heart and great vessels; Ventricular septal defect 1; Thiel-Behnke corneal dystrophy; Thoracic aortic aneurysms and aortic dissections; Marfanoid habitus; Three M syndrome 2; Thrombocytopenia, platelet dysfunction, hemolysis, and imbalanced globin synthesis;

Thrombocytopenia, X-linked; Thrombophilia, hereditary, due to protein C deficiency, autosomal dominant and recessive; Thyroid agenesis; Thyroid cancer, follicular; Thyroid hormone metabolism, abnormal; Thyroid hormone resistance, generalized, autosomal dominant; Thyrotoxic periodic paralysis and Thyrotoxic periodic paralysis 2; Thyrotropin releasing hormone resistance, generalized; Timothy syndrome; TNF receptor-associated periodic fever syndrome (TRAPS); Tooth agenesis, selective, 3 and 4; Torsades de pointes; Townes-Brocks-branchiootorenal-like syndrome; Transient bullous dermolysis of the newborn; Treacher collins syndrome 1; Trichomegaly with mental retardation, dwarfism and pigmentary degeneration of retina; Trichorhinophalangeal dysplasia type I;

Trichorhinophalangeal syndrome type 3; Trimethylaminuria; Tuberous sclerosis syndrome; Lymphangiomyomatosis; Tuberous sclerosis 1 and 2; Tyrosinase-negative oculocutaneous albinism; Tyrosinase-positive oculocutaneous albinism; Tyrosinemia type I; UDPglucose-4- epimerase deficiency; Ullrich congenital muscular dystrophy; Ulna and fibula absence of with severe limb deficiency; Upshaw-Schulman syndrome; Urocanate hydratase deficiency; Usher syndrome, types 1, IB, ID, 1G, 2A, 2C, and 2D; Retinitis pigmentosa 39; UV- sensitive syndrome; Van der Woude syndrome; Van Maldergem syndrome 2; Hennekam lymphangiectasia-lymphedema syndrome 2; Variegate porphyria; Ventriculomegaly with cystic kidney disease; Verheij syndrome; Very long chain acyl-CoA dehydrogenase deficiency; Vesicoureteral reflux 8; Visceral heterotaxy 5, autosomal; Visceral myopathy; Vitamin D-dependent rickets, types land 2; Vi tel 1i form dystrophy ; von Willebrand disease type 2M and type 3; Waardenburg syndrome type 1, 4C, and 2E (with neurologic involvement); Klein-Waardenberg syndrome; Walker-Warburg congenital muscular dystrophy; Warburg micro syndrome 2 and 4; Warts, hypogammaglobulinemia, infections, and myelokathexis; Weaver syndrome; Weill-Marchesani syndrome 1 and 3; Weill- Marchesani-like syndrome; Weissenbacher-Zweymuller syndrome; Werdnig-Hoffmann disease; Charcot-Marie-Tooth disease; Wemer syndrome; WFSl-Related Disorders;

Wiedemann- Steiner syndrome; Wilson disease; Wolfram-like syndrome, autosomal dominant; Worth disease; Van Buchem disease type 2; Xeroderma pigmentosum, complementation group b, group D, group E, and group G; X-linked agammaglobulinemia; X-linked hereditary motor and sensory neuropathy; X-linked ichthyosis with steryl-sulfatase deficiency; X-linked periventricular heterotopia; Oto-palato-digital syndrome, type I; X- linked severe combined immunodeficiency; Zimmermann-Laband syndrome and

Zimmermann-Laband syndrome 2; and Zonular pulverulent cataract 3.

[0344] In some aspects, the present disclosure provides uses of any one of the fusion proteins described herein and a guide RNA targeting this fusion protein to a target T: A base pair in a nucleic acid molecule in the manufacture of a kit for nucleic acid editing, wherein the nucleic acid editing comprises contacting the nucleic acid molecule with the fusion protein and guide RNA under conditions suitable for the substitution of the thymine (T) of the T:A nucleobase pair with an adenine (A). In some embodiments of these uses, the nucleic acid molecule is a double-stranded DNA molecule. In some embodiments, the step of contacting of induces separation of the double- stranded DNA at a target region. In some embodiments, the step of contacting further comprises nicking one strand of the double-stranded DNA, wherein the one strand comprises an unmutated strand that comprises the A of the target T: A nucleobase pair.

[0345] In some embodiments of the described uses, the step of contacting is performed in vitro. In other embodiments, the step of contacting is performed in vivo. In some

embodiments, the step of contacting is performed in a subject (e.g., a human subject or a non human animal subject). In some embodiments, the step of contacting is performed in a cell, such as a human or non-human animal cell.

[0346] The present disclosure also provides uses of any one of the fusion proteins described herein as a medicament. The present disclosure also provides uses of any one of the complexes of fusion proteins and guide RNAs described herein as a medicament.

Pharmaceutical compositions

[0347] Other embodiments of the present disclosure relate to pharmaceutical compositions comprising any of the fusion proteins or the fusion protein-gRNA complexes described herein. The term“pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).

[0348] In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some

embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments pharmaceutical composition comprises a gRNA, a napDNAbp-dCas9 fusion protein, and a pharmaceutically acceptable excipient. In some embodiments pharmaceutical composition comprises a gRNA, a napDNAbp-nCas9 fusion protein, and a pharmaceutically acceptable excipient. Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

[0349] In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with a any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts. Modification of

pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

[0350] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. [0351] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21^st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. See also PCT application PCT/US2010/055131 (Publication No. WO/2011053982), filed Nov. 2, 2010, incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a nuclease. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the

pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0352] As used here, the term“pharmaceutically acceptable carrier” means a

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

Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose,

methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants may also be present in the formulation. The terms such as“excipient”,“carrier”,“pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0353] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and

intracerebroventricular administration.

[0354] In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site. In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

[0355] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. [0356] The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in“stabilized plasmid- lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

[0357] The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term“unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0358] Further, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container containing a compound of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0359] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the disclosure. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution.

It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Delivery Methods

[0360] In some embodiments, the disclosure provides methods comprising delivering any of the fusion proteins, gRNAs, and/or complexes described herein. In other embodiments, the disclosure provides methods comprising delivery of one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some embodiments, the disclosure further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.

[0361] Conventional viral and non-viral based gene transfer methods may be used to introduce nucleic acids in mammalian cells or target tissues. Such methods may be used to administer nucleic acids encoding components of a base editor to cells in culture, or in a host organism. Non-viral vector delivery systems include ribonucleoprotein (RNP) complexes, DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIB TECH 11:211-217 (1993); Mitani & Caskey,

TIB TECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149- 1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0362] In certain embodiments, the method of delivery and vector provided herein is an RNP complex. RNP delivery of base editors markedly increases the DNA specificity of base editing. RNP delivery of base editors leads to decoupling of on- and off-target editing. RNP delivery ablated off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery, and greatly reduced off-target editing even at the highly repetitive VEGFA site 2. See Rees, H.A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790 (2017), which is incorporated by reference herein in its entirety.

[0363] Methods of non-viral delivery of nucleic acids include RNP complexes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent- enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g.,

Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO

1991/17424; WO 1991/16024. Delivery can be to cells (e.g. in vitro or ex vivo

administration) or target tissues (e.g. in vivo administration).

[0364] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,

Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0365] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors may be administered directly to patients {in vivo) or they may be used to treat cells in vitro, and the modified cells may optionally be administered to patients {ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0366] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al, J. Virol. 65:2220-2224 (1991);

PCT/US 94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.

4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al, J. Virol. 63:03822-3828 (1989).

[0367] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and y2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. Reference is made to US 2003/0087817, published May 8, 2003, International Patent Application No. WO 2016/205764, published December 22, 2016, International Patent Application No. WO 2018/071868, published April 19, 2018, and U.S. Patent Publication No. 2018/0127780, published May 10, 2018, the disclosures of each of which are incorporated herein by reference.

[0368] In various embodiments, the disclosed expression constructs may be engineered for delivery in one or more rAAV vectors. An rAAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). An rAAV may comprise a genetic load (i.e., a recombinant nucleic acid vector that expresses a gene of interest, such as a whole or split fusion protein that is carried by the rAAV into a cell) that is to be delivered to a cell. An rAAV may be chimeric.

[0369] As used herein, the serotype of an rAAV refers to the serotype of the capsid proteins of the recombinant vims. Non-limiting examples of derivatives and pseudotypes include rAAV2/l, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. A non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins is rAAV2/5-lVPlu, which has the genome of AAV2, capsid backbone of AAV5 and VPlu of AAV1. Other non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins are rAAV2/5-8VPlu, rAAV2/9-lVPlu, and rAAV2/9-8VPlu.

[0370] AAV derivatives/pseudotypes, and methods of producing such

derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662- 7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158- 167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001). [0371] Methods of making or packaging rAAV particles are known in the art and reagents are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the rAAV particle can be packaged and subsequently purified.

[0372] In some embodiments, the fusion proteins can be divided at a split site and provided as two halves of a whole/complete fusion protein. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete fusion protein through the self-splicing action of the inteins on each fusion protein half. Split intein sequences can be engineered into each of the halves of the encoded fusion protein to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning TABE.

[0373] These split intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding fusion proteins is larger than the recombinant AAV (rAAV) packaging limit, and so requires different solutions. One such solution is formulating the editor fused to split intein pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional editor protein. Several other special considerations to account for the unique features of base editing are described, including the optimization of second-site nicking targets and properly packaging fusion proteins into virus vectors, including lentiviruses and rAAV.

[0374] Accordingly, the disclosure provides dual rAAV vectors and dual rAAV vector particles that comprise expression constructs that encode two halves of any of the disclosed fusion proteins, wherein the encoded fusion protein is divided between the two halves at a split site. In some embodiments, the two halves may be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete fusion protein through the self-splicing action of the inteins on each fusion protein half. Split intein sequences can be engineered into each of the halves of the encoded fusion protein to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning TABE. [0375] In various embodiments, the fusion proteins may be engineered as two half proteins (i.e., an TABE N-terminal half and a TABE C-terminal half) by“splitting” the whole fusion protein as a“split site.” The“split site” refers to the location of insertion of split intein sequences (i.e., the N intein and the C intein) between two adjacent amino acid residues in the fusion protein. More specifically, the“split site” refers to the location of dividing the whole fusion protein into two separate halves, wherein in each halve is fused at the split site to either the N intein or the C intein motifs. The split site can be at any suitable location in the fusion protein fusion protein, but preferably the split site is located at a position that allows for the formation of two half proteins which are appropriately sized for delivery (e.g., by expression vector) and wherein the inteins, which are fused to each half protein at the split site termini, are available to sufficiently interact with one another when one half protein contacts the other half protein inside the cell.

[0376] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003/0087817, incorporated herein by reference.

[0377] It should be appreciated that any fusion protein, e.g., any of the fusion proteins provided herein, may be introduced into the cell in any suitable way, either stably or transiently. In some embodiments, a fusion protein may be transfected into the cell. In some embodiments, the cell may be transduced or transfected with a nucleic acid construct that encodes a fusion protein. For example, a cell may be transduced (e.g., with a vims encoding a fusion protein), or transfected (e.g., with a plasmid encoding a fusion protein) with a nucleic acid that encodes a fusion protein, or the translated fusion protein. Such transduction may be a stable or transient transduction. In some embodiments, cells expressing a fusion protein or containing a fusion protein may be transduced or transfected with one or more gRNA molecules, for example when the fusion protein comprises a Cas9 (e.g., nCas9) domain. In some embodiments, a plasmid expressing a fusion protein may be introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction or other methods known to those of skill in the art.

Kits and Cells

[0378] This disclosure provides kits comprising a nucleic acid construct comprising nucleotide sequences encoding the fusion proteins, gRNAs, and/or complexes described herein. Some embodiments of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding a thymine alkyltransferase-napDNAbp fusion protein capable of alkylating a thymine in a nucleic acid molecule. In some embodiments, the nucleotide sequence encodes any of the thymine alkyltransferases provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the thymine alkyltransferase.

[0379] Some embodiments of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to a thymine alkyltransferase, or a fusion protein comprising a napDNAbp (e.g., Cas9 domain) and a thymine alkyltransferase as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide nucleic acid backbone, e.g., a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide nucleic acid, e.g., guide RNA backbone. In some embodiments, the kit further comprises an expression construct comprising a nucleotide sequence encoding an iDAR.

[0380] The disclosure further provides kits comprising a fusion protein as provided herein, a gRNA having complementarity to a target sequence, and one or more of the following:

cofactor proteins, buffers, media, and target cells (e.g. human cells). Kits may comprise combinations of several or all of the aforementioned components.

[0381] Some embodiments of this disclosure provide cells comprising any of the fusion proteins or complexes provided herein. In some embodiments, the cells comprise nucleotide constructs that encodes any of the fusion proteins provided herein. In some embodiments, the cells comprise any of the nucleotides or vectors provided herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art.

[0382] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL- 2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H- 10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT- 29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYOl, LNCap, Ma-Mel 1-48, MC- 38, MCF-7, MCF-IOA, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI- H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC)

(Manassus, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.

EXAMPLES

Example 1. An RsmE Base Editor

[0383] Alkylation of a targeted thymine to N3-methyl thymine or N3-carboxymethyl thymine, each of which disrupts existing hydrogen bonding with the adenine of the unmutated strand, may be catalyzed by a fusion protein. Without wishing to be bound by any particular theory, the cell’s replication machinery interprets the alkylated thymine as an A, and converts the mismatched adenine of the non-edited strand to a thymine (FIG. 1A). E. coli RsmE has been reported to alkylate uridine at the N3 position within rRNA.

[0384] E. coli RsmE was purified and isolated. The RsmE was tethered to a dCas9 using a SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5) linker. The fusion protein was introduced to E. coli cells. The RsmE protein was sequenced by LC-MS/MS. The RsmE gene was cloned and the activity of the encoded protein confirmed.

Example 2. Evolving the RsmE Base Editor

[0385] Following protein purification and sequencing, variants of RsmE are evolved using PACE systems to form a large library of RsmE mutants. Mutants are cloned into a vector coding for an N-terminal fusion with a dCas9. Mutants are then subjected to selection based on ability to convert thymine into N3-methyl-thymine in DNA using an exemplary antibiotic resistance selection, such as spectinomycin selection system.

[0386] For example, the E. coli selection strain is transformed with a) an accessory plasmid containing an RsmE mutant-dCas9 fusion and targeting guide RNAs, and b) a selection plasmid containing an inactivated spectinomycin resistance gene with a mutation at the active site (D182V) that requires T:A to A:T editing to correct (FIG. 3). Cells harboring RsmE mutants that restore antibiotic resistance are isolated and subjected to additional successive rounds of mutation and selection under varying selection stringencies.

[0387] Those RsmE variants that confer a survival advantage to E. coli cells containing the edited selection gene of > 100-fold are tested for base editing activity in human and murine cells. If N3 -methyl-thymine excision by the cell’s native repair machinery limits editing efficiency, the methylated thymine can be protected from base excision repair by fusing to the candidate T-to-A base editor (TABE) to a known iDAR (e.g., a TDG inhibitor, MBD4 inhibitor, or inhibitor of an AlkbH enzyme, or the catalytically inactive versions of these enzymes) that retains a native ability to tightly bind N3-methyl-thymine-containing DNA. See, e.g., Norman, D. P., Chung, S. J. & Verdine, G. L., Structural and biochemical exploration of a critical amino acid in human 8-oxo-guanine glycosylase, Biochemistry 42, 1564-1572 (2003), and Banerjee, A., Santos, W. L. & Verdine, G. L., Structure of a DNA glycosylase searching for lesions, Science 311, 1153-1157 (2006), the disclosures of each of which are incorporated by reference herein in their entireties.

[0388] Candidate TABEs are characterized in human (HEK293T) and murine cell lines across > 30 endogenous genomic loci to assess editing efficiency, product purity, the size of the editing window, and sequence context preferences (FIG. 3). Successive rounds of directed evolution are then performed until the resulting evolved TABEs perform at a level useful to the genome editing community (e.g. > 20% editing, > 50% product purity, < 5% indels, and an editing window of 2-8 nucleotides). Similar to studies reported with previous base editors, off-target analysis is performed for candidate TABEs at Cas9 nuclease off- targets identified by GUIDE-seq or EndoV-Seq using the same sgRNAs. See Tsai, S. Q. el al, GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nature Biotechnology 33, 187-197 (2015) and Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67 (2019), each of which is incorporated herein in its entirety.

[0389] If RsmE ultimately proves unsuccessful, selections and evolutions are performed using other candidate N3-methyl-thymine-generating enzymes that are known to alkylate pyridines at N3. These enzymes include, but are not limited to, Saccharomyces cerevisiae Bmt5, Saccharomyces cerevisiae Bmt6, human Tsr3, Saccharomyces cerevisiae Tsr3, Vulcaniseta distributa Tsr3, and Sulfolobus solfataricus Tsr3, human DNMT3a, human DNMT3b, human DNMT3L, Hhal methyltransferase from Haemophilus parahaemolyticus, or CpG Methyltransferase (M.SssI) from Spiroplasma monobiae.

EQUIVALENTS AND SCOPE

[0390] In the claims articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0391] Furthermore, the disclosure encompasses all variations, combinations, and

permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or embodiments of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or embodiments of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms“comprising” and“containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0392] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the present disclosure, the

specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0393] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMS What is claimed is:

1. A fusion protein comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) a thymine alkyltransferase.

2. The fusion protein of claim 1, wherein the thymine alkyltransferase methylates thymine to N3-methylthymine.

3. The fusion protein of claim 1, wherein the thymine alkyltransferase alkylates thymine to N3-carboxymethylthymine.

4. The fusion protein of any one of claims 1-3, wherein the thymine alkyltransferase is a wild-type thymine alkyltransferase, or a variant thereof.

5. The fusion protein of any one of claims 1-4, wherein the thymine alkyltransferase is selected from the group consisting of a RsmE, a Bmt5, a Bmt6, a DNMT3a, a DNMT3b, a DNTMT3L, a CpG Methyltransferase (M.SssI), and an Hhal methyltransferase, or a variant thereof.

6. The fusion protein of any one of claims 1-5, wherein the thymine alkyltransferase is a Escherichia coli RsmE, or a variant thereof.

7. The fusion protein of any one of claims 1-5, wherein the thymine alkyltransferase is a Saccharomyces cerevisiae Bmt5, or a variant thereof.

8. The fusion protein of any one of claims 1-5, wherein the thymine alkyltransferase is a Saccharomyces cerevisiae Bmt6, or a variant thereof.

9. The fusion protein of any one of claims 1-8, wherein the thymine alkyltransferase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 51-65.

10. The fusion protein of any one of claims 1-9, wherein the thymine alkyltransferase comprises any one of the amino acid sequences of SEQ ID NOs: 51-65.

11. The fusion protein of any one of claims 4-10, wherein the variant of the wild-type thymine alkyltransferase is produced by evolving a alkyltransferase enzyme.

12. The fusion protein of claim 11, wherein the evolving includes phage assisted continuous evolution (PACE).

13. The fusion protein of claim 11, wherein the evolving includes phage assisted non- continuous evolution (PANCE).

14. The fusion protein of any one of claims 11-13, wherein the variant of the wild-type thymine alkyltransferase is evolved to accept a carboxy-S-adenosyl methionine (carboxy- SAM) cofactor.

15. The fusion protein of any one of claims 1-14, further comprising an inhibitor of DNA alkylation repair (iDAR).

16. The fusion protein of any one of claims 1-15, wherein the fusion protein comprises the structure NH2-[napDNAbp]- [thymine alkyltransferase] -COOH, or NH2-[thymine alkyltransferase]-[napDNAbp]-COOH, wherein each instance of“]-[” indicates the presence of an optional linker sequence.

17. The fusion protein of claim 16, wherein the napDNAbp and the thymine

alkyltransferase are fused via a linker comprising the amino acid sequence.

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

18. The fusion protein of any one of claims 15-17, wherein the fusion protein comprises the structure

Nth- [iDAR] - [napDNAbp] - [thymine alkyltransferase] -COOH;

N¾- [napDNAbp] - [iDAR] - [thymine alkyltransferase] -COOH;

N¾- [napDNAbp] - [thymine alkyltransferase] - [iDAR] -COOH; Nth- [iDAR] -[thymine alkyltransferase] -[napDNAbp] -COOH;

NH2-[thymine alkyltransferase]-[iDAR]-[napDNAbp]-COOH; or

NH2-[thymine alkyltransferase]-[napDNAbp]-[iDAR]-COOH, wherein each instance of

indicates the presence of an optional linker sequence.

19. The fusion protein of claim 18, wherein the napDNAbp and the thymine

alkyltransferase are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

20. The fusion protein of claim 18 or 19, wherein the napDNAbp and the iDAR are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SETPGTSESATPES (SEQ ID NO: 49).

21. The fusion protein of any one of claims 18-20, wherein the thymine alkyltransferase and the iDAR are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

22. A fusion protein comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) an aminocarboxypropyl transferase.

23. The fusion protein of claim 22, wherein the aminocarboxypropyl transferase attaches a 3-amino-3-carboxypropyl group to an N3 position of thymine.

24. The fusion protein of claim 22 or 23, wherein the aminocarboxypropyl transferase is a wild-type aminocarboxypropyl transferase, or a variant thereof.

25. The fusion protein of any one of claims 22-24, wherein the aminocarboxypropyl transferase is a Tsr3 enzyme, or a variant thereof.

26. The fusion protein of any one of claims 22-25, wherein the aminocarboxypropyl transferase is a Homo sapien Tsr3 enzyme, or a variant thereof.

27. The fusion protein of any one of claims 22-25, wherein the aminocarboxypropyl transferase is a Saccharomyces cerevisiae Tsr3 enzyme, or a variant thereof.

28. The fusion protein of any one of claims 22-25, wherein the aminocarboxypropyl transferase is a Vulcanisaeta distributa Tsr3 enzyme, or a variant thereof.

29. The fusion protein of any one of claims 22-25, wherein the aminocarboxypropyl transferase is a Sulfolobus solfataricus Tsr3 enzyme, or a variant thereof.

30. The fusion protein of any one of claims 22-29, wherein the aminocarboxypropyl transferase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 62-65.

31. The fusion protein of any one of claims 22-30, wherein the aminocarboxypropyl transferase comprises any one of the amino acid sequences of SEQ ID NOs: 62-65.

32. The fusion protein of any one of claims 24-31, wherein the variant of the wild-type aminocarboxypropyl transferase is produced by evolving an aminocarboxypropyl transferase enzyme.

33. The fusion protein of claim 32, wherein the variant of the wild-type

aminocarboxypropyl transferase is produced by evolving a Tsr3 enzyme.

34. The fusion protein of claim 32 or 33, wherein the evolving comprises phage assisted continuous evolution (PACE).

35. The fusion protein of claim 32 or 33, wherein the evolving comprises phage assisted non-continuous evolution (PANCE).

36. The fusion protein of any one of claims 22-35 further comprising an inhibitor of DNA alkylation repair (iDAR).

37. The fusion protein of any one of claims 22-36, wherein the fusion protein comprises the structure NH2-[napDNAbp]-[aminocarboxypropyl transferase] -COOH, or N¾- [aminocarboxypropyl transferase]-[napDNAbp]-COOH, wherein each instance of

indicates the presence of an optional linker sequence.

38. The fusion protein of claim 37, wherein the napDNAbp and the aminocarboxypropyl transferase are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

39. The fusion protein of any one of claims 36-38, wherein the fusion protein comprises the structure

NH2-[iDAR]-[napDNAbp]-[aminocarboxypropyl transferase] -COOH;

N¾- [napDNAbp] - [iD AR] - [aminocarboxypropyl transferase] -COOH;

N¾- [napDNAbp] - [aminocarboxypropyl transferase] - [iD AR] -COOH;

N¾- [iD AR] - [aminocarboxypropyl transferase] - [napDNAbp] -COOH;

N¾- [aminocarboxypropyl transferase] -[iDAR]- [napDNAbp] -COOH; or

N¾- [aminocarboxypropyl transferase] -[napDNAbp] -[iDAR] -COOH, wherein each instance of“]-[” indicates the presence of an optional linker sequence.

40. The fusion protein of claim 39, wherein the napDNAbp and the aminocarboxypropyl transferase are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

41. The fusion protein of claim 39 or 40, wherein the napDNAbp and the iDAR are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

42. The fusion protein of any one of claims 39-41, wherein the aminocarboxypropyl transferase and the iDAR are fused via a linker comprising the amino acid sequence

SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 5), GGG, GGGS (SEQ ID NO: 10), SGGGS (SEQ ID NO: 1), or SGSETPGTSESATPES (SEQ ID NO: 49).

43. The fusion protein of any one of claims 1-42, wherein the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9, a CasX, a CasY, a Cpfl, a C2cl, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Casl2a, a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, an LbCasl2a, an AsCasl2a, a Cas9-KKH, a circularly permuted Cas9, an Argonaute (Ago), a SmacCas9, or a Spy-macCas9 domain.

44. The fusion protein of claim 43, wherein the Cas9 domain is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.

45. The fusion protein of claim 43, wherein the Cas9 domain is a nuclease dead Cas9 (dCas9).

46. The fusion protein of claim 43, wherein the Cas9 domain is a Cas9 nickase (nCas9).

47. The fusion protein of claim 43, wherein the Cas9 domain is a nuclease active Cas9.

48. A polynucleotide encoding the fusion protein of any one of claims 1-47.

49. A vector comprising the polynucleotide of claim 48.

50. The vector of claim 49, wherein the vector comprises a heterologous promoter driving expression of the polynucleotide.

51. A complex comprising the fusion protein of any one of claims 1-47 and a guide RNA bound to the nucleic acid programmable DNA binding protein (napDNAbp) of the fusion protein.

52. A cell comprising the fusion protein of any one of claims 1-47, the polynucleotide of claim 48, the vector of claim 49 or 50, or the complex of claim 51.

53. A pharmaceutical composition comprising: (i) the fusion protein of any one of claims 1-47, the polynucleotide of claim 48, the vector of claim 49 or 50, or the complex of claim 51; and

(ii) a pharmaceutically acceptable excipient.

54. A kit comprising a nucleic acid construct, comprising

(i) a nucleic acid sequence encoding the fusion protein of any one of claims 1-47 and a guide RNA; and

(ii) a heterologous promoter that drives expression of the sequence of (i).

55. The kit of claim 54, further comprising an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone

56. A kit comprising:

(i) the fusion protein of any one of claims 1-47;

(ii) a gRNA; and

(iii) target cells.

57. A method for editing a nucleobase pair of a double- stranded DNA sequence, the method comprising:

contacting a double stranded DNA sequence with a complex comprising the fusion protein of any one of claims 1-21 and a guide nucleic acid, wherein the double-stranded DNA comprises a target thymine (T) of an A:T nucleobase pair.

58. The method of claim 57, whereby the thymine (T) is alkylated to N3-methylthymine (m3T).

59. The method of claim 57, whereby the thymine (T) is alkylated to N3- carboxymethylthymine (Cm3T).

60. The method of any one of claims 57-59, whereby the step of contacting induces separation of the double- stranded DNA at a target region.

61. The method of any one of claims 57-60, whereby one strand of the double- stranded DNA is cut, wherein the one strand comprises the adenine (A) of the target A:T nucleobase pair.

62. The method of any one of claims 57-61, whereby the A of the target A:T nucleobase pair is replaced with a thymine (T).

63. The method of any one of claims 57-62, whereby the N3-methylthymine (m3T), or the N3-carboxymethylthymine (Cm3T) is replaced with an adenine (A), thereby generating a T to A point mutation.

64. A method for editing a nucleobase pair of a double- stranded DNA sequence, the method comprising:

comprising contacting the double- stranded DNA sequence with a complex comprising the fusion protein of any one of claims 22-42 and a guide nucleic acid, wherein the double- stranded DNA comprises a target thymine (T) of an A:T nucleobase pair; whereby the thymine is alkylated to an N3-3-amino-3-carboxypropyl thymine.

65. The method of claim 64, whereby the contacting induces separation of the double- stranded DNA at a target region.

66. The method of claim 64 or 65, whereby one strand of the double- stranded DNA is cut, wherein the one strand comprises the A of the target A:T nucleobase pair.

67. The method of any one of claims 64-66, whereby the A of the target A:T nucleobase pair is replaced with a thymine (T).

68. The method of any one of claims 64-67, whereby the N3 3-amino-3-carboxypropyl thymine is replaced with an adenine (A), thereby generating a T to A point mutation.

69. The method of any one of claims 57-68, wherein the method is performed in vitro , in vivo, or ex vivo.

70. The method of any one of claims 57-69, wherein the double- stranded DNA is in a subject.

71. The method of claim 70, wherein the subject is human.

72. A method of treating a subject having or at risk of developing a disease, disorder, or condition, the method comprising:

administering to the subject the fusion protein of any one of claims 1-47, the polynucleotide of claim 48, the vector of claim 49 or 50, the complex of claim 51, or the pharmaceutical composition of claim 53.

73. The method of claim 72, wherein the subject has been diagnosed with a disease, disorder, or condition.

74. The method of claim 72 or 73, wherein the subject has a T to A, or an A to T mutation that is associated with a disease, disorder, or condition.

75. The method of claim 74, wherein the T of the A to T mutation is converted to an A.

76. The method of claim 74 or 75, wherein the A of the T to A mutation is converted to a T.

77. The method of claim 76, wherein the disease, disorder, or condition is sickle cell anemia, Fanconi anemia, ectodermal dysplasia skin fragility syndrome, lattice corneal dystrophy Type III, or Noonan syndrome.

78. The fusion protein of any one of claims 1-5, wherein the fusion protein comprises a human DNMT3a, a human DNMT3b, or a human DNTMT3L, or a variant thereof.

79. The fusion protein of any one of claims 1-5, wherein the fusion protein comprises a CpG Methyltransferase (M.SssI), or a variant thereof.

80. The fusion protein of any one of claims 1-5, wherein the fusion protein comprises an Hhal methyltransferase, or a variant thereof.

81. The fusion protein of any one of claims 1-47, wherein the fusion protein does not comprise an E. coli DNA adenine methyltransferase (Dam), or a variant thereof.

82. The fusion protein of any one of claims 1-47, wherein the fusion protein does not comprise a DNA (cytosine-5)-methyltransferase 1 (DNMT1).

83. Use of (a) a fusion protein of any one of claims 1-47 or 78-82 and (b) a guide RNA targeting the base editor of (a) to a target A:T nucleobase pair in a double- stranded DNA molecule in DNA editing.

84. The use of claim 83, whereby the DNA editing comprises nicking one strand of the double-stranded DNA, wherein the one strand comprises the A of the target T: A nucleobase pair.

85. Use of a fusion protein of any one of claims 1-47 or 78-82, or a complex of claim 51, as a medicament.