BACKGROUND OF THE INVENTION

Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the 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 human genetic diseases.1An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency of installing the desired modification; (2) minimal off-target activity; and (3) the ability to be programmed to edit precisely any site in a given nucleic acid, e.g., any site within the human genome.2Current genome engineering tools, including engineered zinc finger nucleases (ZFNs),3transcription activator like effector nucleases (TALENs),4and most recently, the RNA-guided DNA endonuclease Cas9,5effect sequence-specific DNA cleavage in a genome. This programmable cleavage can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR).6,7

One drawback to the current technologies is that both NHEJ and HDR are stochastic processes that typically result in modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration.8Since many genetic diseases in principle can be treated by effecting a specific nucleotide change at a specific location in the genome (for example, a C to T change in a specific codon of a gene associated with a disease),9the development of a programmable way to achieve such precision gene editing would represent both a powerful new research tool, as well as a potential new approach to gene editing-based human therapeutics.

SUMMARY OF THE INVENTION

Nucleic acid programmable DNA binding proteins (napDNAbp), such as the clustered regularly interspaced short palindromic repeat (CRISPR) system is a recently discovered prokaryotic adaptive immune system10that has been modified to enable robust and general genome engineering in a variety of organisms and cell lines.11CRISPR-Cas (CRISPR associated) systems are protein-RNA complexes that use an RNA molecule (sgRNA) as a guide to localize the complex to a target DNA sequence via base-pairing.12In the natural systems, a Cas protein then acts as an endonuclease to cleave the targeted DNA sequence.13The target DNA sequence must be both complementary to the sgRNA, and also contain a “protospacer-adjacent motif” (PAM) at the 3′-end of the complementary region in order for the system to function.14

Among the known Cas proteins,S. pyogenesCas9 has been mostly widely used as a tool for genome engineering.15This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind DNA in a sgRNA-programmed manner.16In principle, when fused to another protein or domain, dCas9 can target that protein or domain to virtually any DNA sequence simply by co-expression with an appropriate sgRNA.

The potential of the dCas9 complex for genome engineering purposes is immense. Its unique ability to bring proteins to specific sites in a genome programmed by the sgRNA in theory can be developed into a variety of site-specific genome engineering tools beyond nucleases, including deaminases (e.g., cytidine deamianses), transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases.11Some of these potential applications have recently been implemented through dCas9 fusions with transcriptional activators to afford RNA-guided transcriptional activators,17,18transcriptional repressors, 16,19,20 and chromatin modification enzymes.21Simple co-expression of these fusions with a variety of sgRNAs results in specific expression of the target genes. These seminal studies have paved the way for the design and construction of readily programmable sequence-specific effectors for the precise manipulation of genomes.

Some aspects of the disclosure are based on the recognition that certain configurations of a nucleic acid programmable DNA binding protein (napDNAbp), for example CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with a cytidine deaminase domain fused to the N-terminus of a napDNAbp via a linker was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. See, for example, Examples 3 and 4 below, which demonstrate that the fusion proteins, which are also referred to herein as base editors, generate less indels and more efficiently deaminate target nucleic acids than other base editors, such as base editors without a UGI domain. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with a cytidine deaminase domain fused to the N-terminus of napDNAbp via a linker perform base editing with higher efficiency and greatly improved product purity when the fusion protein is comprised of more than one UGI domain. See, for example, Example 17, which demonstrates that a fusion protein (e.g., base editor) comprising two UGI domains generates less indels and more efficiently deaminates target nucleic acids than other base editors, such as those comprising one UGI domain.

In some embodiments, the fusion protein comprises: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; and (iii) a uracil glycosylase inhibitor (UGI) domain, where the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein. In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a CasX protein. In some embodiments, the CasX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 or 30. In some embodiments, the CasX protein comprises the amino acid sequence of SEQ ID NO: 29 or 30.

In some embodiments, the fusion protein comprises: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; (iii) a first uracil glycosylase inhibitor (UGI) domain; and (iv) a second uracil glycosylase inhibitor (UGI) domain, wherein the napDNAbp is a Cas9, dCas9, or Cas9 nickase protein. In some embodiments, the napDNAbp is a dCas9 protein. In some embodiments, the napDNAbp is a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein. In some embodiments, the dCas9 protein is aS. pyogenesdCas9 (SpCas9d). In some embodiments, the dCas9 protein is aS. pyogenesdCas9 harboring a D10A mutation. In some embodiments, the dCas9 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 6 or 7. In some embodiments, the dCas9 protein comprises the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the dCas9 protein is aS. aureusdCas9 (SaCas9d). In some embodiments, the dCas9 protein is aS. aureusdCas9 harboring a D10A mutation. In some embodiments, the dCas9 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 33-36. In some embodiments, the dCas9 protein comprises the amino acid sequence of SEQ ID NO: 33-36.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a CasY protein. In some embodiments, the CasY protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 31. In some embodiments, the CasY protein comprises the amino acid sequence of SEQ ID NO: 31.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cpf1 or Cpf1 mutant protein. In some embodiments, the Cpf1 or Cpf1 mutant protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 9-24. In some embodiments, the Cpf1 or Cpf1 mutant protein comprises the amino acid sequence of any one of SEQ ID NOs: 9-24.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a C2c1 protein. In some embodiments, the C2c1 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26. In some embodiments, the C2c1 protein comprises the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a C2c2 protein. In some embodiments, the C2c2 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27. In some embodiments, the C2c2 protein comprises the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a C2c3 protein. In some embodiments, the C2c3 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28. In some embodiments, the C2c3 protein comprises the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is an Argonaute protein. In some embodiments, the Argonaute protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the Argonaute protein comprises the amino acid sequence of SEQ ID NO: 25.

Some aspects of the disclosure are based on the recognition that fusion proteins provided herein are capable of generating one or more mutations (e.g., a C to T mutation) without generating a large proportion of indels. In some embodiments, any of the fusion proteins (e.g., base editing proteins) provided herein generate less than 10% indels. In some embodiments, any of the fusion proteins (e.g., base editing proteins) provided herein generate less than 10%, 9%, 8%, 7%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% indels.

In some embodiments, the fusion protein comprises a napDNAbp and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the napDNAbp comprises the amino acid sequence of any of the napDNAbp provided herein. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 76). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 74). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 81). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 82-84). In some embodiments, the fusion protein comprises a napDNAbp and an apolipoprotein B mRNA-editing complex 1 catalytic polypeptide-like 3G (APOBEC3G) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker of any length or composition (e.g., an amino acid sequence, a peptide, a polymer, or a bond). In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605).

In some embodiments, the fusion protein comprises a napDNAbp and a cytidine deaminase 1 (CDA1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605). In some embodiments, the napDNAbp comprises the amino acid sequence of any of the napDNAbps provided herein.

In some embodiments, the fusion protein comprises a napDNAbp and an activation-induced cytidine deaminase (AID) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605). In some embodiments, the napDNAbp comprises the amino acid sequence of any of the napDNAbps provided herein.

Some aspects of the disclosure are based on the recognition that certain configurations of a napDNAbp, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain fused to the N-terminus of a napDNAbp via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604) was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. In some embodiments, the fusion protein comprises a napDNAbp domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the napDNAbp via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604).

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within a subject's genome, e.g., a human's genome. In some embodiments, fusion proteins of napDNAbp (e.g., CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein) and deaminases or deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of napDNAbp and deaminases or deaminase domains, are provided.

Some aspects of this disclosure provide fusion proteins comprising a napDNAbp as provided herein that is fused to a second protein (e.g., an enzymatic domain such as a cytidine deaminase domain), thus forming a fusion protein. In some embodiments, the second protein comprises an enzymatic domain, or a binding domain. In some embodiments, the enzymatic domain is a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments, the enzymatic domain is a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). It should be appreciated that the deaminase may be from any suitable organism (e.g., a human or a rat). In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 76). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 74). In some embodiments, the deaminase is pmCDA1.

Some aspects of this disclosure provide fusion proteins comprising: (i) a CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein domain comprising the amino acid sequence of SEQ ID NO: 32; and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the napDNAbp via a linker comprising the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 76). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 74). In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 5737. In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 81). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a human APOBEC3G variant of any one of SEQ ID NOs: 82-84.

Other aspects of this disclosure relate to the recognition that fusion proteins comprising a deaminase domain, a napDNAbp domain and a uracil glycosylase inhibitor (UGI) domain demonstrate improved efficiency for deaminating target nucleotides in a nucleic acid molecule. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for a decrease in nucleobase editing efficiency in cells. Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated herein, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Without wishing to be bound by any particular theory, base excision repair may be inhibited by molecules that bind the single strand, block the edited base, inhibit UGI, inhibit base excision repair, protect the edited base, and/or promote “fixing” of the non-edited strand, etc. Thus, this disclosure contemplates fusion proteins comprising a napDNAbp-cytidine deaminase domain that is fused to a UGI domain.

Further aspects of this disclosure relate to the recognition that fusion proteins comprising a deaminase domain, a napDNAbp domain, and more than one uracil glycosylase inhibitor (UGI) domain (e.g., one, two, three, four, five, or more UGI domains) demonstrate improved efficiency for deaminating target nucleotides in a nucleic acid molecule and/or improved nucleic acid product purity. Without wishing to be bound by any particular theory, the addition of a second UGI domain may substantially decrease the access of UDG to the G:U base editing intermediate, thereby improving the efficiency of the base editing.

Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base 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 base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels.

In certain embodiments, any of the base editors provided herein are capable of generating a certain percentage of desired mutations. In some embodiments, the desired mutation is a C to T mutation. In some embodiments, the desired mutation is a C to A mutation, In some embodiments, the desired mutation is a C to G mutation. In some embodiments, any of the base editors provided herein are capable of generating at least 1% of desired mutations. In some embodiments, any of the base editors provided herein are capable of generating at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of desired mutations.

Some aspects of the disclosure are based on the recognition that any of the base editors 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, the deaminase domain of the fusion protein is fused to the N-terminus of the napDNAbp domain. In some embodiments, the UGI domain is fused to the C-terminus of the napDNAbp domain. In some embodiments, the napDNAbp and the nucleic acid editing domain are fused via a linker. In some embodiments, the napDNAbp domain and the UGI domain are fused via a linker. In some embodiments, a second UGI domain is fused to the C-terminus of a first UGI domain. In some embodiments, the first UGI domain and the second UGI domain are fused via a linker.

In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the invention. 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 heteroaliphatic 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 moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (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 moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included functionalized moieties 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.

In some embodiments, the linker comprises the amino acid sequence (GGGGS)n(SEQ ID NO: 607), (G)n(SEQ ID NO: 608), (EAAAK)n(SEQ ID NO: 609), (GGS)n(SEQ ID NO:610), (SGGS)n(SEQ ID NO: 606), SGSETPGTSESATPES (SEQ ID NO: 604), (XP)n(SEQ ID NO: 611), SGGS(GGS)n(SEQ ID NO: 612), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605), or any combination thereof, wherein n is independently an integer between 1 and 30, and X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n(SEQ ID NO: 610), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGGS(GGS)n(SEQ ID NO: 612), wherein n is 2. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 604). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 605).

In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[napDNAbp]-[optional linker sequence]-[UGI]. In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[UGI]-[optional linker sequence]-[napDNAbp]; [UGI]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[napDNAbp]; [UGI]-[optional linker sequence]-[napDNAbp]-[optional linker sequence]-[nucleic acid editing domain]; [napDNAbp]-[optional linker sequence]-[UGI]-[optional linker sequence]-[nucleic acid editing domain]; [napDNAbp]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[UGI]; or [nucleic acid editing domain]-[optional linker sequence]-[napDNAbp]-[optional linker sequence]-[first UGI]-[optional linker sequence]-[second UGI].

In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a cytidine deaminase 1 (CDA1). In some embodiments, the deaminase is a Lamprey CDA1 (pmCDA1) deaminase.

In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat. In some embodiments, the deaminase is a rat APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 76). In some embodiments, the deaminase is a human APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 74). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 81). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 60). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 82-84). In some embodiments, the deaminase is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 49-84.

In some embodiments, the UGI domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 134. In some embodiments, the UGI domain comprises the amino acid sequence as set forth in SEQ ID NO: 134.

Some aspects of this disclosure provide complexes comprising a napDNAbp fusion protein as provided herein, and a guide RNA bound to the napDNAbp.

Some aspects of this disclosure provide methods of using the napDNAbp, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with a napDNAbp or a fusion protein as provided herein and with a guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence; or (b) with a napDNAbp, a napDNAbp fusion protein, or a napDNAbp or napDNAbp complex with a gRNA as provided herein.

Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp or a napDNAbp fusion protein 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 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.

Some aspects of this disclosure provide polynucleotides encoding a napDNAbp of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide.

Some aspects of this disclosure provide cells comprising a napDNAbp protein, a fusion protein, a nucleic acid molecule, and/or a vector as provided herein.

It should be appreciated that any of the fusion proteins provided herein that include a Cas9 domain (e.g. Cas9, nCas9, or dCas9) may be replaced with any of the napDNAbp provided herein, for example CasX, CasY, Cpf1, C2c1, C2c2, C2c3, or Argonaute protein.

The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

