Nucleic acid binding domains and methods of use thereof

Abstract

Provided herein are polypeptides, compositions comprising the polypeptides and methods for genome editing and gene regulation (e.g., activation and / or repression) using the polypeptides or the compositions comprising the polypeptides, such as, DNA binding domains derived from the genus of Legionella. These DNA binding proteins include a fragment of N-cap sequence and / or a C-cap sequence of a DNA binding protein derived from the genus of Legionella.

Description

NUCLEIC ACID BINDING DOMAINS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to United States Provisional Application Serial No. 63 / 518,221 filed August 8, 2023, the disclosure of which is herein incorporated in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING XML

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “ALTI- 740WO_SEQ_LIST” created on August 6, 2024, and having a size of 82,066 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.INTRODUCTION

[0003] Genome editing and gene regulation techniques include the use of nucleic acid binding proteins. These proteins may be used directly to alter chromatin structure and / or modulate binding of transcriptional regulators or may be linked to a heterologous protein for gene editing or modulating gene expression.

[0004] Provided herein are polypeptides and methods of use thereof for genome modification such as genome editing and gene regulation. These polypeptides include nucleic acid binding domain derived from DNA binding proteins from bacteria from the genus of Legionella.SUMMARY

[0005] In various aspects, the present disclosure provides a polypeptide that binds to a target nucleic acid sequence and includes an N-cap region, a plurality of repeat units (RUs) that mediates specific binding to the target nucleic acid sequence, and a C-cap region. The N-cap region may have a length that is shorter than the length of the N-cap region of a DNA binding protein from Legionella quateirensis (L. quaieirensis) or Legionella maceachemii (L. maceachernii). The C-cap region may have a length that is shorter than the length of the C-cap region of a DNA binding protein from L. quateirensis or L. maceachernii. The plurality of RUs may include RUs derived from the DNA binding protein from L. quateirensis or L. maceachernii.

[0006] In certain embodiments, the N-cap region of the polypeptide that binds to the target nucleic acid sequence is at least 119 amino acids long and comprises at least residues N119 to N1 ofthe N-cap region of the DNA binding protein from L. quateirensis, where the N-cap region does not include residues N143 to N120 of the N-cap region of the DNA binding protein from L. quateirensis, where residue N143 refers to the N-terminus amino acid (i.e., the 1stresidue) and residue N1 refers to the C-terminus amino acid (i.e., the last residue) of the full-length N-cap region of the DNA binding protein from L. quateirensis. In certain embodiments, the N-cap region of the polypeptide may not include at least amino acids N143 to N93 of the N-cap region of the DNA binding protein from L. quateirensis. In certain embodiments, the N-cap region may not include at least amino acids N143 to N68 of the N-cap region of the DNA binding protein from L. quateirensis. In certain embodiments, the N-cap region may not include at least amino acids N143 to N44 of the N-cap region of the DNA binding protein from L. quateirensis. In certain embodiments, the N-cap region may not include at least amino acids N143 to N25 of the N-cap region of the DNA binding protein from L. quateirensis. The N-terminus region of the polypeptide may include residues N1 to up to N119, residues N1 to up to N92, residues N1 to up to N67, residues N1 to up to N43, or residues N1 to up to N24 of the N-cap region of the DNA binding protein from L. quateirensis. The C-cap region may include full length C-cap region of the DNA binding protein from L. quateirensis or a fragment thereof.

[0007] In certain embodiments, the C-cap region of the polypeptide that binds to the target nucleic acid sequence is truncated relative to the full-length C-cap region of the Legionella quateirensis DNA-binding polypeptide, wherein the full-length C-cap region extends from residue Cl to residue C162, where residue Cl refers to the N-terminus (i.e., the 1stresidue) and residue C162 refers to the C-terminus amino acid (i.e., the last residue) of the full-length C-cap region of the Legionella quateirensis DNA-binding polypeptide. In certain embodiments, the C-cap region is truncated to a position from C136 to C17 and includes at least residues from Cl to C17 to up to Cl to Cl 36 relative to the C-cap region of the Legionella quateirensis DNA-binding polypeptide. In certain embodiments, the C-cap region is a fragment of the C-cap region of the Legionella quateirensis DNA-binding polypeptide and includes residues Cl to Cl 36. The N-cap region may include full length N-cap region of the DNA binding protein from L. quateirensis or a fragment thereof, such as the shorter N-cap regions provided herein.

[0008] In certain aspects, the DNA binding protein from L. quateirensis has an amino acid sequence as set forth in SEQ ID NO:1. In certain aspects, the N-cap region of the DNA binding protein from L. quateirensis has an amino acid sequence as set forth in SEQ ID NO:2. In certainaspects, the C-cap region of the DNA binding protein from L. quateirensis has an amino acid sequence as set forth in SEQ ID NO:3.

[0009] In certain embodiments, the N-cap region of the polypeptide that binds to the target nucleic acid sequence comprises at least residues N85 to N1 of the N-cap region of the DNA binding protein from L. maceachemii, where the N-cap region does not include residues N99 to N86 of the N-cap region of the DNA binding protein from L. maceachemii, where residue N99 refers to the N- terminus amino acid (i.e., the 1stresidue) and residue N1 refers to the C-terminus amino acid (i.e., the last residue) of the full-length N-cap region of the DNA binding protein from L. maceachemii. In certain embodiments, the N-cap region may not include at least amino acids N99 to N63 of the N- cap region of the DNA binding protein from L. maceachemii. In certain embodiments, the N-cap region may not include at least amino acids N99 to N50 of the N-cap region of the DNA binding protein from L. maceachemii. In certain embodiments, the N-cap region of the polypeptide may not include at least amino acids N99 to N23 of the N-cap region of the DNA binding protein from L. maceachemii. In certain embodiments, the N-tcrminus region of the polypeptide may include residues N1 to up to N85, residues N1 to up to N62, residues N1 to up to N49, or residues N1 to up to N22 of the N-cap region of the DNA binding protein from L. maceachemii. The C-cap region may include full length C-cap region of the DNA binding protein from L. maceachemii or a fragment thereof, such as, a fragment as described herein.

[0010] In certain embodiments, the C-cap region of the polypeptide that binds to the target nucleic acid sequence is truncated relative to the full-length C-cap region of the Legionella maceachemii DNA-binding polypeptide, wherein the full-length C-cap region extends from position Cl to position C313. In certain embodiments, the C-cap region is truncated to a position from C269 to C30. In certain embodiments, the C-cap region is truncated to position C269. The N-cap region may include full length N-cap region of the DNA binding protein from L. maceachemii or a fragment thereof. room In certain aspects, the DNA binding protein from L. maceachemii has an amino acid sequence as set forth in SEQ ID NO:4. In certain aspects, the N-cap region of the DNA binding protein from L. maceachemii has an amino acid sequence as set forth in SEQ ID NO:5. In certain aspects, the C-cap region of the DNA binding protein from L. maceachemii has an amino acid sequence as set forth in SEQ ID NO:6.

[0012] Additional embodiments are described below.INCORPORATION BY REFERENCE

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF DRAWINGS

[0014] FIG. 1A, from top to bottom, shows a schematic of a DNA binding protein of L. quateirensis', a DNA binding protein of L. maceachernii', a DNA binding protein of L. quateirensis with a FLAG tag and a nuclear localization sequences (NLS) at the N-terminus, and a Human influenza hemagglutinin (HA) tag at the C-terminus; and a DNA binding protein of L. maceachernii with a FLAG tag and a NLS at the N-terminus, and a HA tag at the C-terminus. The proteins are not drawn to scale. The DNA binding protein of L. quateirensis binds to a nucleic acid comprising a sequence of G / AAGTGTCTCG (SEQ ID NO: 14). The DNA binding protein of L. maceachernii binds to a nucleic acid comprising a sequence of GTCAGGACTCT (SEQ ID NO: 15).

[0015] FIG. IB provides a schematic of a DNA binding protein with an N-cap region of the DNA binding protein from L. quateirensis, RUs for binding a target nucleotide sequence, and a C- cap region of the DNA binding protein from L. quateirensis.

[0016] FIGS. 1C-1D provide schematics of a DNA binding protein with a truncated N-cap region of the DNA binding protein from L. quateirensis, RUs for binding a target nucleotide sequence, and a truncated C-cap region of the DNA binding protein from L. quateirensis.

[0017] FIG. IE provides a schematic of a DNA binding protein with an N-cap region of the DNA binding protein from L. maceachernii, RUs for binding a target nucleotide sequence, and a C- cap region of the DNA binding protein from L. maceachernii.

[0018] FIGS. 1F-1G provide schematics of a DNA binding protein with a truncated N-cap region of the DNA binding protein from L. maceachernii, RUs for binding a target nucleotide sequence, and a truncated C-cap region of the DNA binding protein from L. maceachernii.

[0019] FIG. 2 provides a schematic of DNA binding proteins of L. quateirensis and L. maceachernii with various deletions in the N-cap region and / or the C-cap region and the experimental workflow for analysis of the proteins.

[0020] FIGS. 3 and 4 show a Western blot of the DNA binding proteins (DBPs) as depicted in Fig. 2. An anti-FLAG tag antibody was used to detect the DBPs after electrophoretic separation of proteins from a complex mixture of IVTT reactions.

[0021] FIG. 5 shows the structure of LEF104 and TEF10 proteins. LEF104 protein includes the wild-type DNA binding protein of L. quateirensis and protein domains, FLAG-tag and nuclear localization sequence (NLS) added to the N-terminus and protein domains, HA and WPRE, added at the C-terminus. TEF10 protein which includes a truncated C-cap region which is 17 amino acid long and does not include residue C18 to C162 relative to the 162 amino acids long C-cap region of the wild-type DNA binding protein of L. quateirensis which includes residue Cl to Cl 62. Both the wildtype DNA binding protein of L. quateirensis LEF104 and the TEF10 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14), as determined by DNA-protein binding in SELEX.

[0022] FIG. 6 shows the structure of LEF104 and TEF17 proteins. TEF17 protein includes an N-cap region deletion (deletion of the lsL24 amino acids) and a C-cap region deletion (deletion of the last 145 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region, respectively, of DNA binding protein of L. quateirensis. Both the wild-type DNA binding protein of L. quateirensis LEF104 and the TEF17 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0023] FIG. 7 shows the structure of TEF24 protein which includes an N-cap region deletion (deletion of the 1st51 amino acids) and a C-cap region deletion (deletion of the last 145 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis. Both the wild-type DNA binding protein of L. quateirensis and the TEF24 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0024] FIG. 8 shows the structure of TEF16 protein which includes an N-cap region deletion (deletion of the 1st24 amino acids)and a C-cap region deletion (deletion of the last 132 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis. Both the wild-type DNA binding protein of L. quateirensis and the TEF16 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0025] FIG. 9 shows the structure of TEF21 protein which includes an N-cap region deletion (deletion of the 1st51 amino acids)and a C-cap region deletion (deletion of the last 80 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis. Both the wild-type DNA binding protein of L. quateirensis and the TEF21 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0026] FIG. 10 shows the structure of TEF22 protein which includes an N-cap region deletion (deletion of the 1st51 amino acids)and a C-cap region deletion (deletion of the last 99 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap regionof DNA binding protein of L. quateirensis . Both the wild-type DNA binding protein of L. quateirensis and the TEF22 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0027] FIG. 11 shows the structure of TEF23 protein which includes an N-cap region deletion (deletion of the 1st51 amino acids)and a C-cap region deletion (deletion of the last 132 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis. Both the wild- type DNA binding protein of L. quateirensis and the TEF23 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0028] FIG. 12 shows the structure of TEF27 protein which includes an N-cap region deletion (deletion of the 1st76 amino acids)and a C-cap region deletion (deletion of the last 29 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis. Both the wild-type DNA binding protein of L. quateirensis and the TEF27 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14).

[0029] FIG. 13 shows the structure of TEF28 protein which includes an N-cap region deletion (deletion of the 1st76 acids)and a C-cap region deletion (deletion of the last 80 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis. Both the wild-type DNA binding protein of L. quateirensis and the TEF28 protein bind to the sequence of G / AAGTGTCTCG (SEQ ID NO: 14). LEF104 refers to the wild-type DNA binding protein of L. quateirensis with the tags as depicted in the schematic.

[0030] FIG. 14 shows the structure of TEF40 protein which includes an N-cap region deletion (deletion of the 1st119 acids)and a C-cap region deletion (deletion of the last 26 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis and its binding to DNA (SEQ ID NO: 14).

[0031] FIG. 15 shows the structure of TEF41 protein which includes an N-cap region deletion (deletion of the 1st119 acids)and a C-cap region deletion (deletion of the last 49 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis and its binding to DNA.

[0032] FIG. 16 shows the structure of TEF42 protein which includes an N-cap region deletion (deletion of the 1st119 acids)and a C-cap region deletion (deletion of the last 80 amino acids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis and its binding to DNA (SEQ ID NO: 14).

[0033] FIG. 17 shows the structure of TEF44 protein which includes an N-cap region deletion (deletion of the 1st119 acids)and a C-cap region deletion (deletion of the last 132 aminoacids) relative to the 143 amino acids long N-cap region and the 162 amino acids long C-cap region of DNA binding protein of L. quateirensis and its binding to DNA (SEQ ID NO: 14).

[0034] FIG. 18 provides a comparison of TEF28, TEF42, and TEF44 binding to LEF104 (SEQ ID NO: 14).

[0035] FIG. 19 summarizes the results obtained from the various N-cap, C-cap, or both deletion versions of the DNA binding protein of L. quateirensis. TEF5-TEF9, TEF11-TEF15, TEF18-TEF20, TEF25-TEF26, TEF30, TEF32-TEF-39 and TEF43 were not tested. TEF10, TEF16- TEF17, TEF21-TEF24, and TEF27-TEF28 showed binding to the expected DNA sequence.

[0036] FIG. 20 shows the structure of TEF46 protein which includes the N-cap region of the DNA binding protein of L. maceachernii and a C-cap region deletion (deletion of the last 44 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachernii. LEF70 refers to the wild-type DNA binding protein “LEF70” from L. maceachernii with the tags as depicted. The LEF70 protein binds to the sequence GTCAGGACTCT (SEQ ID NO: 15).

[0037] FIG. 21 shows the structure of TEF47 protein which includes the N-cap region of the DNA binding protein of L. maceachernii and a C-cap region deletion (deletion of the last 76 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachernii and its binding to DNA (SEQ ID NO: 15).

[0038] FIG. 22 shows the structure of TEF49 protein which includes the N-cap region of the DNA binding protein of L. maceachernii and a C-cap region deletion (deletion of the last 155 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachernii and its binding to DNA.

[0039] FIG. 23 shows the structure of TEF50 protein which includes the N-cap region of the DNA binding protein of L. maceachernii and a C-cap region deletion (deletion of the last 184 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachernii and its binding to DNA.

[0040] FIG. 24 shows the structure of TEF51 protein which includes the N-cap region of the DNA binding protein of L. maceachernii and a C-cap region deletion (deletion of the last 206 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachernii and its binding to DNA.

[0041] FIG. 25 shows the structure of TEF52 protein which includes the N-cap region of the DNA binding protein of L. maceachernii and a C-cap region deletion (deletion of the last 233 aminoacids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachemii and its binding to DNA.

[0042] FIG. 26 shows the structure of TEF53 protein which includes the N-cap region of the DNA binding protein of L. maceachemii and a C-cap region deletion (deletion of the last 251 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachemii and its binding to DNA.

[0043] FIG. 27 shows the structure of TEF54 protein which includes the N-cap region of the DNA binding protein of L. maceachemii and a C-cap region deletion (deletion of the last 283 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachemii and its binding to DNA.

[0044] FIG. 28 shows the structure of TEF73 protein which includes the N-cap region deletion (deletion of the 1st37 amino acids) relative to the N-cap region of the DNA binding protein of L. maceachemii and a C-cap region deletion (deletion of the last 251 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachemii and its binding to DNA.

[0045] FIG. 29 shows the structure of TEF82 protein which includes the N-cap region deletion (deletion of the 1st50 amino acids) relative to the N-cap region of the DNA binding protein of L. maceachemii and a C-cap region deletion (deletion of the last 283 amino acids) relative to the 313 amino acids long C-cap region of DNA binding protein of L. maceachemii and its binding to DNA.

[0046] FIG. 30 shows results of ELISA assay with the truncated L. maceachemii proteins.

[0047] FIG. 31 summarizes the results obtained from the various N-cap, C-cap, or both deletion versions of the DNA binding protein of L. maceachemii. Crossed-out constructs were not tested.DETAILED DESCRIPTION

[0048] In various aspects, the present disclosure provides a polypeptide that includes an N- cap region, a plurality of repeat units (RUs) that mediates specific binding to a target nucleic acid sequence, and a C-cap region. The N-cap region may have a length that is shorter than the length of the N-cap region of a DNA binding protein from L. quateirensis or L. maceachemii. The C-cap region may have a length that is shorter than the length of the C-cap region of a DNA binding protein from L. quateirensis or L. maceachemii. The sequences of the repeat units may be derivedfrom a DNA binding protein from Legionella. These sequences can be linked together to form non- naturally occurring modular nucleic acid binding domains (NBDs), capable of targeting and binding any target nucleic acid sequence (e.g., DNA sequence).Definitions

[0049] As used herein, the term “derived” in the context of a polypeptide thereof indicates that the polypeptide includes an amino acid sequence of a protein that is from a particular source (e.g., Legionella), that is a variant of the protein from a particular source (e.g., Legionella), is a mutated or modified form of the protein from a particular source (e.g., Legionella), and shares at least 30% sequence identity with, at least 40% sequence identity with, at least 50% sequence identity with, at least 60% sequence identity with, at least 70% sequence identity with, at least 80% sequence identity with, or at least 90% sequence identity with a protein from a particular source (e.g., Legionella).

[0050] As used herein, the term “modular” indicates that a particular polypeptide such as a nucleic acid binding domain, comprises a plurality of repeat units that can be switched and replaced with other repeat units. For example, any repeat unit in a modular nucleic acid binding domain can be switched with a different repeat unit. In some embodiments, modularity of the nucleic acid binding domains disclosed herein allows for switching the target nucleic acid base for a particular repeat unit by simply switching it out for another repeat unit. In some embodiments, modularity of the nucleic acid binding domains disclosed herein allows for swapping out a particular repeat unit for another repeat unit to increase the affinity of the repeat unit for a particular target nucleic acid. Overall, the modular nature of the nucleic acid binding domains disclosed herein enables the development of genome editing complexes that can precisely target any nucleic acid sequence of interest.

[0051] In particular embodiments, modular nucleic acid binding domains (NBDs), also referred to herein as “DNA binding polypeptides,” are provided herein from the genus of Legionella. In some embodiments, modular nucleic acid binding domains derived from Legionella can be engineered to bind to a target gene of interest for purposes of gene editing or gene regulation. The nucleic acid sequence can be DNA or RNA.

[0052] The terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids,and polypeptides having modified polypeptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminus methionine residues; immunologically tagged proteins; and the like. In specific embodiments, the terms refer to a polymeric form of amino acids of any length which include genetically coded amino acids. In particular embodiments, the terms refer to a polymeric form of amino acids of any length which include genetically coded amino acids fused to a heterologous amino acid sequence.

[0053] The term “heterologous” refers to two components that are defined by structures derived from different sources. For example, in the context of a polypeptide, a “heterologous” polypeptide may include operably linked amino acid sequences that are derived from different polypeptides (e.g., a NBD and a functional domain derived from different sources). Similarly, in the context of a polynucleotide encoding a chimeric polypeptide, a “heterologous” polynucleotide may include operably linked nucleic acid sequences that can be derived from different genes. Other exemplary “heterologous” nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin than the promoter, the coding sequence or both). In the context of recombinant cells, “heterologous” can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present.

[0054] The term “operably linked” refers to linkage between molecules to provide a desired function. For example, “operably linked” in the context of nucleic acids refers to a functional linkage between nucleic acid sequences. By way of example, a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) may be operably linked to a second polynucleotide, wherein the expression control sequence affects transcription and / or translation of the second polynucleotide. In the context of a polypeptide, “operably linked” refers to a functional linkage between amino acid sequences (e.g., different domains) to provide for a described activity of the polypeptide.

[0055] As used herein, the term “cleavage” refers to the breakage of the covalent backbone of a nucleic acid, e.g., a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single- stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as aresult of two distinct single- stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, the polypeptides provided herein are used for targeted double- stranded DNA cleavage.

[0056] A “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).

[0057] A “target nucleic acid,” “target sequence,” or “target site” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule, such as, the NBD disclosed herein will bind. The target nucleic acid may be present in an isolated form or inside a cell. A target nucleic acid may be present in a region of interest. A “region of interest” may be any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and / or targeted recombination, targeted activation or repression. A region of interest can be present in a chromosome, an cpisomc, an organcllar genome (c.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, promoter sequences, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.

[0058] An “exogenous” molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule, e.g., a gene or a gene segment lacking a mutation present in the endogenous gene. An exogenous nucleic acid can be present in an infecting viral genome, a plasmid or episome introduced into a cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co -precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.

[0059] By contrast, an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplastor other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.

[0060] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control region.

[0061] “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, shRNA, RNAi, miRNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs which arc modified, by processes such as capping, polyadcnylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP- ribosylation, myristylation, and glycosylation.

[0062] ‘Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, donor integration, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a polypeptide or has not been modified by a polypeptide as described herein. Thus, gene inactivation may be partial or complete.

[0063] The terms “patient” or “subject” are used interchangeably to refer to a human or a non-human animal (e.g., a mammal).

[0064] The terms “treat”, “treating”, treatment” and the like refer to a course of action (such as administering a polypeptide comprising a NBD fused to a heterologous functional domain or a nucleic acid encoding the polypeptide) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.

[0065] The terms “prevent”, “preventing”, “prevention” and the like refer to a course of action (such as administering a polypeptide comprising a NBD fused to a heterologous functional domain or a nucleic acid encoding the polypeptide) initiated in a manner (e.g., prior to the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject’s risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof, generally in the context of a subject predisposed to having a particular disease, disorder or condition. In certain instances, the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.

[0066] The phrase “therapeutically effective amount” refers to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient. The therapeutically effective amount can be ascertained by measuring relevant physiological effects.

[0067] Percent identity between a pair of sequences may be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region, including gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. Only internal gaps are included in the length, not gaps at the sequence ends.

[0068] Percent Identity = (Matches x 100) / Length of aligned region (with gaps)

[0069] The phrase “conservative amino acid substitution” refers to substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Conservative amino acid substitutions may preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.

[0070] Guidance for substitutions, insertions, or deletions may be based on alignments of amino acid sequences of proteins from different species or from a consensus sequence based on a plurality of proteins having the same or similar function.Nucleic Acid Binding Polypeptides

[0071] The present disclosure provides a polypeptide that specifically binds to a target nucleic acid sequence and includes an N-cap region, a plurality of repeat units, wherein each repeat unit recognizes and binds to a single nucleotide (in DNA or RNA), and a C-cap region. Each repeat unit in the plurality of repeat units can be specifically selected to target and bind to a specific nucleic acid sequence. The N-cap region and the C-cap region is included to provide additional binding functionality by, e.g., increasing specificity and / or affinity for a target sequence. A N-cap region and / or the C-cap region is truncated to decrease overall size of the polypeptide while preserving the binding specificity of the polypeptide.

[0072] In certain aspects, the N-cap region of the polypeptides disclosed herein may have a deletion of at least 24 amino acids at the N-terminus relative to the full-length N-cap region of the DNA binding protein of L. quateirensis. In certain aspects, the N-cap region may have a deletion of at least 51 amino acids, at least 76 amino acids, at least 100 amino acids, or at least 119 amino acids, or up to 119 amino acids at the N-terminus relative to the full-length N-cap region of the DNA binding protein of L. quateirensis. The N-cap region may have a deletion of at least 24 amino acids at the N-terminus relative to the full-length N-cap region of the DNA binding protein of L. quateirensis and may be at least 24 amino acids in length. The N-cap region may have a deletion of at least 24 amino acids at the N-terminus relative to the full-length N-cap region of the DNA binding protein of L. quateirensis and may be up to 119 amino acids long, up to 92 amino acids long, up to 67 amino acids long, up to 43 amino acids long, or up to 24 amino acids long. The N-cap region may have a deletion of at least 24 amino acids at the N-terminus relative to the full-length N-cap region of the DNA binding protein of L. quateirensis and may be up to 119 amino acids long, at least 92 amino acids long, at least 67 amino acids long, at least 43 amino acids long, or at least 24 amino acids long. In certain aspects, the C-cap region may not have any deletions and may be of the same length as the C-cap region of the DNA binding protein of L. quateirensis.

[0073] In certain aspects, the C-cap region of the polypeptide may have a deletion of at least 26 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. quateirensis. In certain aspects, the C-cap region of the polypeptide may have a deletion of at least 26 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. quateirensis and may be at least 17 amino acids long. In certain aspects, the C-cap region may have a deletion of at least 49 amino acids, at least 80 amino acids, at least 99 amino acids, at least 132 amino acids, or at least 145 amino acids, or up to 145 amino acids at the C- terminus relative to the full-length C-cap region of the DNA binding protein of L. quateirensis . TheC-cap region may have a deletion of at least 26 amino acids at the C-terminus relative to the full- length C-cap region of the DNA binding protein of L. quateirensis and may be up to 136 amino acids long, up to 113 amino acids long, up to 82 amino acids long, up to 63 amino acids long, up to 30 amino acids long, or up to 17 amino acids long. The C-cap region may have a deletion of at least 26 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. quateirensis and may be up to 136 amino acids long, at least 113 amino acids long, at least 82 amino acids long, at least 63 amino acids long, at least 30 amino acids long, or at least 17 amino acids long. In certain aspects, the N-cap region may not have any deletions and may be of the same length as the N-cap region of the DNA binding protein of L. quateirensis.

[0074] The DNA binding protein of L. quateirensis may have a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the following sequence:

[0075] MPDLELNFAIPLHLFDDETVFTHDATNDNSQASSSYSSKSSPASANARKRTSR KEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLI LYKGRTSAVQQLLCKHEELLNLISPDGLGHKELIKIAARNGGGNNLIAVLSCYAKLKEMGFS SQQIIRMVSHAGGANNLKAVTANHDDLQNMGFNVEQIVRMVSHNGGSKNLKAVTDNHDD LKNMGFNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMGFNAEQIVSMVSNNGGSKNLK AVTDNHDDLKNMGFNAEQIVSMVSNGGGSLNLKAVKKYHDALKDRGFNTEQIVRMVSHD GGSLNLKAVKKYHDALRERKFNVEQIVSIVSHGGGSLNLKAVKKYHDVLKDREFNAEQIV RMVSHDGGSLNLKAVTDNHDDLKNMGFNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFHF TADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSSTPSPSAP SFFQGPSTPIPTPVLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKNFNVNN FWSLFPFDDVGYHPHSNDVGYHLHSDEESPFFDF (SEQ ID NO:1). For example, the DNA binding protein of L. quateirensis may have the sequence set forth in SEQ ID NO:1 other than 1-5, 5-8, 8-10, 10-15, 15-20, 1-18, 1-15, 2-14, 3-13, 4-12, 5-10, or 1-10 conservative amino acid substitutions.

[0076] The full-length N-cap region of the DNA binding protein of L. quateirensis may have a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the following sequence:

[0077] MPDLELNFAIPLHLFDDETVFTHDATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID N0:2) or may have the sequence set forth inSEQ ID N0:2 other than 1-20, 1-18, 1-15, 2-14, 3-13, 4-12, 5-10, or 1-10 conservative amino acid substitutions.

[0078] As used herein, the term “Nl” in the context of a L. quateirensis N-cap region refers to the C-terminus amino acid “G” and the term “N143” refers to the N-terminus amino acid “M” of SEQ ID NO:2.

[0079] The full-length C-cap region of the DNA binding protein of L. quateirensis may have the following sequence:

[0080] FTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSST PSPSAPSFFQGPSTPIPTPVLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKN FNVNNFWSLFPFDDVGYHPHSNDVGYHLHSDEESPFFDF (SEQ ID NO:3) or may have the sequence set forth in SEQ ID NO:3 with 1-20, 1-18, 1-15, 2-14, 3-13, 4-12, 5-10, or 1-10 conservative amino acid substitutions.

[0081] As used herein, the term “Cl” in the context of a L. quateirensis C-cap region refers to the N- tcrminus amino acid “F” and the term “C162” refers to the C-tcrminal amino acid “F” of SEQ ID NO:3.

[0082] In certain embodiments, a truncated N-cap region may include an amino acid sequence that is identical or similar to the amino acid sequence of the corresponding region of the full-length N-cap region. For example, a truncated N-cap region that includes a deletion of at least 24 amino acids at the N-terminus relative to the full-length N-cap region may include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the corresponding sequence of the full-length N-cap region (i.e., to the sequence from Nl 19-N1 of SEQ ID NO:2).

[0083] In certain embodiments, the N-cap region is at least or up to 24 amino acids long and comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of N24-N1 of SEQ ID NO:2 as set forth below:

[0084] GRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO: 18)

[0085] In certain embodiments, a truncated N-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of N43-N1 of SEQ ID NO:2 as set forth below:

[0086] SLLSLN YTKKQIERLILYKGRTS A VQQLLCKHEELLNLISPDG (SEQ ID NO : 19)

[0087] In certain embodiments, a truncated N-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of N67-N1 of SEQ ID NO:2 as set forth below:

[0088] NKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLL CKHEELLNLISPDG (SEQ ID NO:20)

[0089] In certain embodiments, a truncated N-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of N92-N1 of SEQ ID NO:2 as set forth below:

[0090] SRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLS LNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO:21)

[0091] In certain embodiments, a truncated N-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of N119-N1 of SEQ ID NO:2 as set forth below:

[0092] ATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQN NKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLI SPDG (SEQ ID NO:22)

[0093] In certain embodiments, a truncated C-cap region may include an amino acid sequence that is identical or similar to the amino acid sequence of the corresponding region of the full-length C-cap region. For example, a truncated C-cap region that includes a deletion of at least 26 amino acids at the C-terminus relative to the full-length C-cap region may include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the corresponding sequence of the full-length C-cap region (i.e., to the sequence from C1-C136 of SEQ ID NOG).

[0094] In certain embodiments, a truncated C-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of C1-C136 of SEQ ID NOG as set forth below:

[0095] FTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNN TSSSTPSPSAPSFFQGPSTPIPTPVLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDLDSQLG DPTKNFNVNNFWSLFPFDD (SEQ ID NO:23)

[0096] In certain embodiments, a truncated C-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of Cl-Cl 13 of SEQ ID NOG as set forth below:

[0097] FTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNN TSSSTPSPSAPSFFQGPSTPIPTPVLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDL (SEQ ID NO:24)

[0098] In certain embodiments, a truncated C-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of C1-C82 of SEQ ID NOG as set forth below:

[0099] FTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSS TPSPSAPSFFQGPSTPIPTPVLDNS (SEQ ID NO:25)

[0100] In certain embodiments, a truncated C-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of C1-C63 of SEQ ID NOG as set forth below:

[0101] FTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSS TPSPSA (SEQ ID NO:26)

[0102] In certain embodiments, a truncated C-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of C1-C30 of SEQ ID NOG as set forth below:

[0103] FTADQIVALICQSKQCFRNLKKNHQQWKNK (SEQ ID NO:27)

[0104] In certain embodiments, a truncated C-cap region comprises or consists of an amino acid sequence that at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of C1-C17 of SEQ ID NOG as set forth below:

[0105] FTADQIVALICQSKQCF (SEQ ID NO:28)

[0106] The N-cap regions and C-cap regions and fragments thereof derived from the DNA binding protein of L. quateirensis as provided herein may be used in any combination to generate DNA binding polypeptides. The RUs may be derived from any DNA binding protein (DBP), such as, the DBPs described herein.

[0107] In certain aspects, the polypeptide for binding to a target nucleic acid sequence may include a truncated N-cap region relative to the N-cap region of a L. quateirensis DNA-binding polypeptide and a truncated C-cap region relative to the C-cap region of the L. quateirensis DNA- binding polypeptide, where the N-cap region is truncated by to up to 76 amino acids from the N- terminus relative to the N-cap region of the L. quateirensis DNA-binding polypeptide and the N-cap region is truncated by to up to 80 amino acids at the C-terminus relative to the C-cap region of the L. quateirensis DNA-binding polypeptide.

[0108] In certain aspects, the polypeptide for binding to a target nucleic acid sequence may include a truncated N-cap region relative to the N-cap region of a L. quateirensis DNA-binding polypeptide and a truncated C-cap region relative to the C-cap region of the L. quateirensis DNA- binding polypeptide, where the N-cap region is truncated by to up to 51 amino acids from the N- terminus relative to the N-cap region of the L. quateirensis DNA-binding polypeptide and the N-cap region is truncated by to up to 80 amino acids, up to 99 amino acids, up to 132 amino acids, or up to 145 amino acids at the C-terminus relative to the C-cap region of the L. quateirensis DNA-binding polypeptide.

[0109] In certain aspects, the polypeptide for binding to a target nucleic acid sequence may include a truncated N-cap region relative to the N-cap region of a Legionella maceachemii (L. maceachemii) DNA-binding polypeptide and / or a truncated C-cap region relative to the C-cap region of the L. maceachemii DNA-binding polypeptide. In certain embodiments, the N-cap region of the polypeptide may have a deletion of at least 1 amino acids at the N-terminus relative to the full-length N-cap region of a L. maceachemii DNA-binding polypeptide. In certain aspects, the N- cap region may be at least 22 amino acids long. In certain aspects, the N-cap region may have a deletion of at least 37 amino acids at the N-terminus relative to the full-length N-cap region. In certain aspects, the N-cap region may have a deletion of at least 50 amino acids at the N-terminus relative to the full-length N-cap region. In certain aspects, the N-cap region may have a deletion of at least 77 amino acids at the N-terminus relative to the full-length N-cap region. In certain aspects, the N-cap region may have a deletion of up to 77 amino acids at the N-terminus relative to the full- length N-cap region.

[0110] In certain aspects, the C-cap region of the polypeptide may have a deletion of at least 44 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. maceachemii. In certain aspects, the C-cap region of the polypeptide may be at least 30 amino acids in length. In certain aspects, the C-cap region of the polypeptide may be up to 30 amino acids in length. In certain aspects, the C-cap region may have a deletion of at least 76 amino acids, at least 106 amino acids, at least 155 amino acids, at least 184 amino acids, at least 206 amino acids, at least 233 amino acids, at least 251 amino acids, at least 283 amino acids, or up to 283 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. maceachemii.

[0111] The DNA binding protein of L. maceachemii may have a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the following sequence:

[0112] MPKTNQPKNLEAKSTKNKISLPQDPQTLNELKIKGYPQDLAERLIKKGSSLAV KTVLKDHEQLVNFFTHLQIIRMAAQKGGAKNITTALNEYNSLTNLGYSSEQIVRVAAHGGG SLNIKAVLQAHQALKELDFSAEQIVRIAAHDGGSLNIDAVQQAQQALKELGFSAKHIVRIAA HIGGSLNIKAVQQAQQALKELGFSADQIVRIAAHKGGSHNIVAVQQAQQALKELDFSAEQIV RIAAHIGGSRNIEAIQQAHHALKELGFSAEQIVRIAAHIGGSHNLKAVLQAQQALKELDFSAE QIVRIAAHDGGSRNIEAVQQAQHVLKELGFSAEQIVHIAAHGGGSLNIKAILQAHQTLKELN FSAEQIVSIVAHDGGSRNIEAVQQAQHILKELGFSTEQIVCIAGHGGGSLNIKAVLLAQQALK DLGFSAEQIVSIAAHVGGSHNIEAVQKAHQALKELDFSAEQIVRIAAHIGGSRNIEATIKHYA MLTQPPYMLSQEQFLRLIDHHSGHLNLSILLDEQQWQAINDLCLQPHHFGRQNALEKFLQQ GQRKYQNLQELEQFLFQDSADPMLLQETENQHEAEKINDCMDFILRLISATEPLDLQIEIEGI GLFSPSMHFDATQANFSTPAANEEKIDNSATEAGVNSRKRKIAAAHQKQPPRKKTATPLSAT FISTLTTLAQSDNPRLEMASAEALMLKAPQKLAMGITVRKKTKCEGIAIITVTDKTKLNGWL SSASESTYSSVEAQGTRTVNNTHAFFSTPLTSDKKSPSFSSLDFYEDSGLGFDEEITNPPYMPE LEPEFIL (SEQ ID NO:4). For example, the DNA binding protein of L. maceachernii may have the sequence set forth in SEQ ID NO:4 other than 1-5, 5-8, 8-10, 10-15, 15-20, 1-18, 1-15, 2-14, 3-13, 4-12, 5-10, or 1-10 conservative amino acid substitutions.

[0113] The full-length N-cap region of the DNA binding protein of L. maceachernii may have the following sequence may have a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the following sequence:

[0114] MPKTNQPKNLEAKSTKNKISLPQDPQTLNELKIKGYPQDLAERLIKKGSSLAV KTVLKDHEQLVNFFTHLQIIRMAAQKGGAKNITTALNEYNSLTNLG (SEQ ID NO:5) or may have the sequence set forth in SEQ ID NO:5 with 1-5, 5-8, 8-10, 10-15, 15-20, 1-18, 1-15, 2-14, 3- 13, 4-12, 5-10, or 1-10 conservative amino acid substitutions.

[0115] As used herein, the term “Nl” in the context of a L. maceachernii N-cap region refers to the C-terminus amino acid “G” and the term “N99” refers to the N-terminus amino acid “M” of SEQ ID NO:5.

[0116] The full-length C-cap region of the DNA binding protein of L. maceachernii may have a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the following sequence:

[0117] YMLSQEQFLRLIDHHSGHLNLSILLDEQQWQAINDLCLQPHHFGRQNALEKFLQQGQRKYQNLQELEQFLFQDSADPMLLQETENQHEAEKINDCMDFILRLISATEPLDLQIEIEGIGLFSPSMHFDATQANFSTPAANEEKIDNSATEAGVNSRKRKIAAAHQKQPPRKKTATPL SATFISTLTTLAQSDNPRLEMASAEALMLKAPQKLAMGITVRKKTKCEGIAIITVTDKTKLN GWLSSASESTYSSVEAQGTRTVNNTHAFFSTPLTSDKKSPSFSSLDFYEDSGLGFDEEITNPP YMPELEPEFIL (SEQ ID NO:6) or may have the sequence set forth in SEQ ID NO:6 with 1-5, 5-8, 8-10, 10-15, 15-20, 1-18, 1-15, 2-14, 3-13, 4-12, 5-10, or 1-10 conservative amino acid substitutions.

[0118] As used herein, the term “Cl” in the context of a L. maceachemii C-cap region refers to the N-terminal amino acid “Y” and the term “C3I3” refers to the C-terminal amino acid “L” of SEQ ID NO:6.

[0119] The truncated N-cap region may have the same sequence or a similar sequence as the corresponding region in the full-length N-cap region from L. maceachemii. The truncated C-cap region may have the same sequence or a similar sequence as the corresponding region in the full- length C-cap region from L. maceachemii.

[0120] The isolated polypeptides disclosed herein may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or more, e.g., up to 45 repeat units. In certain embodiments, the isolated polypeptide may include at least 9 RUs. In certain embodiments, the isolated polypeptide may include at least 10 RUs. In certain embodiments, the isolated polypeptide may include at least 11 RUs. In certain embodiments, the isolated polypeptide may include at least 12 RUs. In certain embodiments, the isolated polypeptide may include RUs in the range of 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, or 19-20 RUs, or any number of RUs in between these ranges.

[0121] In some aspects, the RUs have a sequence of Ai-nXiX2Bi4-33,34,35, wherein: each amino acid residue of AMI is any amino acid residue; X1X2 comprises base-contacting residue(s) (BCR) that determine the base (A, G, T, or C) to which the repeat unit binds, where Xi is any amino acid and X2 is absent or is any amino acid; each amino acid residue of B 14-33,34,35 is any amino acid.

[0122] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FNAEQIVRMVSX1X2GGSLNLKAVTDNHDDLKNMG (SEQ ID NOU), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2 is absent or is any amino acid.

[0123] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence LGHKELIKIAAX1X2GGGNNLIAVLSCYAKLKEMG (SEQ ID NO:65), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid, for example, XiX2=RN.

[0124] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FSSQQIIRMVSX1X2GGANNLKAVTANHDDLQNMG (SEQ ID NO:66), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=HA.

[0125] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FNVEQIVRMVSXIX2GGSKNLKAVTDNHDDLKNMG (SEQ ID NO:67), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=HN.

[0126] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FNAEQIVRMVSX1X2GGSKNLKAVTDNHDDLKNMG (SEQ ID NO:68) or FNVEQIVSIVS XIX2GGSLNLKAVKKYHDVLKDRE (SEQ ID NO:69), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=HG.

[0127] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FNAEQIVSMVSX1X0GGSKNLKAVTDNHDDLKNMG (SEQ ID NO:70), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=NN.

[0128] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequenceFNAEQIVSMVSX1X2GGSLNLKAVKKYHDALKDRG (SEQ ID N0:71), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=NG.

[0129] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FNTEQIVRMVSXIX2GGSLNLKAVKKYHDALRERK (SEQ ID NO:72) or FNAEQIVRMVS X1X2GGSLNLKAVTDNHDDLKNMG (SEQ ID NO:73), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=HD.

[0130] In some aspects, the plurality of RUs comprise at least one RU comprising a 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% identical, or 100% identical to the amino acid sequence FNAEQIVRMVSXIX2GGSKNLALVKEYFPVFSSFH (SEQ ID NO:74), where XiX2is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2is absent or is any amino acid. For example, XiX2=HK.

[0131] In some embodiments, any repeat unit disclosed herein, e.g., a RU comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence FNAEQIVRMVSXIX2GGSLNLKAVTDNHDDLKNMG (SEQ ID NOU) or FNAEQIVRMVSX1X2GGSKNLALVKEYFPVFSSFH (SEQ ID NO:75), can have a BCR selected from HA, HD, HG, HK, HN, ND, NG, NI, NH, NK, NN, NP, NT, QN, RN, RS, SH, SI, or SN. In some embodiments, a RU comprising the BCR HD mediates binding to cytosine. In some embodiments, a RU comprising the BCR NG or HG mediates binding to thymine. In some embodiments, a RU comprising the BCR NI, HI, RN, NT, HA, RS, or SI mediates binding to adenine. In some embodiments, a RU comprising the BCR RN, HN, NH, NN, HK, or NK mediates binding to guanine. In some embodiments, the polypeptides disclosed herein may include an N-cap region that is a truncated relative to the N-cap region of L. maceachernii, a plurality of RUs arranged to bind to a target nucleic acid sequence, and a C-cap region that is a truncated relative to the C-cap region of L. maceachernii, as described herein. In some embodiments, the polypeptides disclosed herein may include an N-cap region that is a truncated relative to the N-cap region of L. quateirensis,a plurality of RUs arranged to bind to a target nucleic acid sequence, and a C-cap region that is a truncated relative to the C-cap region of L. quateirensis, as described herein.

[0132] In some embodiments, a half repeat unit may also be included in the isolated polypeptide at, e.g., as the last repeat unit. In some embodiments, the half repeat unit may comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence FNAEQIVRMVSHKGGSKNL (SEQ ID NO:8) or FNAEQIVRMVSX1X2GGSKNL (SEQ ID NO:76) and Xi is any amino acid and X2is absent or is any amino acid. In some embodiments, XIX2=HA, HD, HG, HK, HN, ND, NG, NI, NH, NK, NN, NP, NT, QN, RN, RS, SH, SI, or SN.

[0133] In some aspects, the plurality of RUs comprise at least one RU comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence FSAEQIVRIAAX1X2GGSLNIKAVQQAQQALKELG (SEQ ID NO:9) Or FSAEQIVRIAAX1X2GGSRNIEATIKHYAMLTQPP (SEQ ID NO:77), where X1X2 is a BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2 is absent or is any amino acid. In some embodiments, a BCR is selected from HA, HD, HG, HK, HN, ND, NG, NI, NH, NK, NN, NP, NT, QN, RN, RS, SH, SI, or SN. In some embodiments, a RU comprising the BCR HD mediates binding to cytosine. In some embodiments, a RU comprising the BCR NG or HG mediates binding to thymine. In some embodiments, a RU comprising the BCR NI, HI, RN, NT, HA, RS, or SI mediates binding to adenine. In some embodiments, a RU comprising the BCR RN, HN, NH, NN, HK, or NK mediates binding to guanine. In some embodiments, the N- cap region is a truncated relative to the N-cap region of L. maceachemii and the C-cap region is a truncated relative to the C-cap region of L. maceachemii, as described herein. In some embodiments, the half repeat unit may comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence FSAEQIVRIAAX1X2GGSRNIEA (SEQ ID NO: 10), wherein is a XiX2BCR(s) that mediates binding to a base (A, G, T, or C) and Xi is any amino acid and X2 is absent or is any amino acid. In some embodiments, a BCR is selected from HA, HD, HG, HK, HN, ND, NG, NI, NH, NK, NN, NP, NT, QN, RN, RS, SH, SI, or SN.

[0134] In some embodiments, the RUs may be those derived from RUs present in Xanthomonas spp TALE proteins. For example, one or more of the plurality of RUs may comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, orat least 99% identical to the amino acid sequence LTPDQVVAIASX12X13GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 11). The half repeat can have 15 to 23 amino acid residues, for example, the half repeat can have 19 amino acid residues. In particular embodiments, the half repeat can have a sequence as set forth in LTPQQVVAIASX12X13GGRPALE (SEQ ID NO: 12). X12X13 can be any amino acid and can be selected based on the nucleotide in the target sequence bound by the RU.

[0135] Truncation of the N-cap and / or the C-cap regions can be particularly advantageous for obtaining DNA binding domains, which are smaller in size. A reduced number of amino acids can allow for more efficient packaging into a viral vector and a smaller molecular weight can result in more efficient loading of the DNA binding domains in non- viral vectors for delivery.

[0136] In some embodiments, the N-cap region may be directly linked to the RUs or may be linked via a spacer sequence. In some embodiments, the repeat unit may be directly linked to each other or may be linked via spacer sequences. In some embodiments, the last repeat unit or half-repeat unit (if present) may be directly linked to the C-cap region or may be linked via spacer sequences. The spacer sequences may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids. The spacer sequence may include amino acids that have small side chains, e.g., alanine, glycine, serine, etc. In certain aspects, the spacer sequence is a flexible linker.

[0137] In certain aspects, the polypeptides disclosed herein may include a nuclear localization sequence (NLS) to facilitate entry into the nucleus of a cell, e.g., an animal or a plant cell. In some embodiments, the NLS may be present at the N-terminus or C-terminus of the polypeptide.

[0138] In certain aspects, the polypeptide may be produced in a host cell and expressed with a translocation signal at the N-terminus which translocation signal may be cleaved during translocation. The translocated protein may be purified from the cell culture medium.Fusion Proteins

[0139] In certain aspects, the polypeptides disclosed herein can be linked to a heterologous protein. The heterologous protein may be linked to the N-terminus or C-terminus of the polypeptides disclosed herein. The heterologous protein may include a functional domain. The functional domaincan provide different activities, such as genome editing, gene regulation (e.g., activation or repression), or visualization of a genomic locus via imaging.A. Genome Editing Domains

[0140] The polypeptides provided herein can be linked to a nuclease, wherein the RUs provide specificity and targeting and the nuclease provides genome editing functionality. In some embodiments, the nuclease can be a cleavage domain, which dimerizes with another copy of the same cleavage domain to form an active full domain capable of cleaving DNA. In other embodiments, the nuclease can be a cleavage domain, which is capable of cleaving DNA without needing to dimerize. For example, a nuclease comprising a cleavage domain can be an endonuclease, such as FokI or Bfil. In some embodiments, two cleavage domains (e.g., FokI or Bfil) can be fused together to form a fully functional single cleavage domain. When cleavage domains are used as the nuclease, two DNA binding polypeptides (DBPs) can be engineered, the first DBP binding to a top strand of a target nucleic acid sequence and comprising a first FokI cleavage domain and a second DBP binding to a bottom strand of a target nucleic acid sequence and comprising a second FokI cleavage domain.

[0141] In some embodiments, a fully functional cleavage domain, capable of cleaving DNA without needing to dimerize include meganucleases, also referred to as homing endonucleases. For example, a meganuclease can include I-Anil or I-Onul. In some embodiments, the nuclease can be a type IIS restriction enzyme, such as FokI or Bfil.

[0142] A nuclease domain fused to a DBP provided herein, can be an endonuclease or an exonuclease. An endonuclease can include restriction endonucleases and homing endonucleases. An endonuclease can also include SI Nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, or yeast HO endonuclease. An exonuclease can include a 3’-5’ exonuclease or a 5’-3’ exonuclease. An exonuclease can also include a DNA exonuclease or an RNA exonuclease. Examples of exonuclease includes exonucleases I, II, III, IV, V, and VIII; DNA polymerase I, RNA exonuclease 2, and the like. The polypeptides disclosed herein are also referred to as DNA binding proteins (DBPs) or DNA binding domains, however, these polypeptides may also bind RNA.

[0143] A nuclease domain fused to a DBP can be a restriction endonuclease (or restriction enzyme). In some instances, a restriction enzyme cleaves DNA at a site removed from the recognition site and has a separate binding and cleavage domains. In some instances, such restriction enzyme is a Type IIS restriction enzyme.

[0144] A nuclease domain fused to a DBP can be a Type IIS nuclease. A Type IIS nuclease can be FokI or Bfil. In some cases, a nuclease domain fused to a DBP is Fokl. In other cases, a nuclease domain fused to a DBP is Bfil.

[0145] Fokl can be a wild-type Fokl or can comprise one or more mutations. In some cases, Fokl can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. A mutation can enhance cleavage efficiency. A mutation can abolish cleavage activity. In some cases, a mutation can modulate homodimerization. For example, Fokl can have a mutation at one or more amino acid residue positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 to modulate homodimerization.

[0146] In some instances, a Fokl cleavage domain is, for example, as described in Kim et al. “Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain,” PNAS 93: 1156-1160 (1996), which is incorporated herein by reference in its entirety. In some cases, a Fokl cleavage domain described herein has a sequence as follows: QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLG GSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWW KVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEE VRRKFNNGEINF (SEQ ID NO: 13). In other instances, a Fokl cleavage domain described herein is a Fokl, for example, as described in U.S. Patent No. 8,586,526, which is incorporated herein by reference in its entirety.

[0147] A DBP can be linked to a functional group that modifies DNA nucleotides, for example an adenosine deaminase.

[0148] For purposes of gene editing, a first DBP linked to a cleavage domain and a second DBP linked to a cleavage domain can be provided. The first DBP linked to a cleavage domain can recognize a top strand of double stranded DNA and bind to said region of double stranded DNA. The second DBP linked to a cleavage domain can recognize a separate, non-overlapping bottom strand of double stranded DNA and bind to said region of double stranded DNA. The target nucleic acid sequence on the bottom strand can have its complementary nucleic acid sequence in the top strand positioned 10 to 20 nucleotides towards the 3’ end from the first region. In some embodiments this stretch of 10 to 20 nucleotides can be referred to as the spacer region. In some embodiments, this first DBP linked to a cleavage domain and the second DBP linked to a cleavage domain both bind at a target site, allowing for dimerization of the two cleavage domains in the spacer region and allowing for catalytic activity and cleaving of the target DNA.B. Regulatory Domains

[0149] As another example, a DBP can be linked to a gene regulating domain. A gene regulation domain can be an activator or a repressor. For example, a DBP can be linked to an activation domain, such as VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldbl self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta). Alternatively, a DBP can be linked to a repressor, such as KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.

[0150] In some embodiments, a DBP can be linked to a DNA modifying protein, such as DNMT3a, a chromatin-modifying protein, such as lysine- specific histone demethylase 1 (LSD1), a protein that is capable of recruiting other proteins, such as KRAB. The DNA modifying protein (e.g., DNMT3a) and proteins capable of recruiting other proteins (e.g., KRAB) can serve as repressors of transcription. Thus, a DBP linked to a DNA modifying protein (e.g., DNMT3a) or a domain capable of recruiting other proteins (e.g., KRAB, a domain found in transcriptional repressors, such as Koxl) can provide gene repression functionality, can serve as transcription factors, wherein the DBP provides specificity and targeting and the DNA modifying protein and the protein capable of recruiting other proteins provides gene repression functionality, which can be referred to as a DBP- transcription factor (DBP-TF).

[0151] In some embodiments, expression of the target gene can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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 92%, at least 95%, at least 97%, or at least 99% by using a DBP- TF of the present disclosure as compared to non-treated cells. In some embodiments, expression of the target gene can be reduced by 5% to 10%, 10% to 15%, 15% to 20%, 20%, to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% by using a DBP-TF of the present disclosure as compared to non-treated cells. In some embodiments, expression of the checkpoint gene can be reduced by over 90% by using a DBP-TF of the present disclosure as compared to non-treated cells.

[0152] In some embodiments, repression of the target gene with a DBP fused to a repression domain (e.g., a DBP-TF) of the present disclosure and subsequent reduced expression of the target gene can last for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, or at least 28 days. In some embodiments, repression of the target gene with the DBP-TF of the present disclosure and subsequent reduced expression of the target gene can last for 1 days to 3 days, 3 days to 5 days, 5 days to 7 days, 7 days to 9 days, 9 days to 11 days, 11 days to 13 days, 13 days to 15 days, 15 days to 17 days, 17 days to 19 days, 19 days to 21 days, 21 days to 23 days, 23 days to 25 days, or 25 days to 28 days.

[0153] In various aspects, the present disclosure provides a method of identifying a target binding site in a target gene of a cell, the method comprising: (a) contacting a cell with an engineered genomic regulatory complex comprising a DBP, a repressor domain, and a linker; (b) measuring expression of the target gene; and (c) determining expression of the target gene is repressed by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% for at least 3 days, wherein the target gene is selected from: a checkpoint gene and a T cell surface receptor.

[0154] In some aspects, expression of the target gene is repressed in at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of a plurality of the cells. In some aspects, the engineered genomic regulatory complex is undetectable after at least 3 days. In some aspects, determining the engineered genomic regulatory complex is undetectable is measured by qPCR, imaging of a FLAG-tag, or a combination thereof. In some aspects, the measuring expression of the target gene comprises flow cytometry quantification of expression of the target gene.

[0155] In some embodiments, repression of the target gene with a DBP-TF of the present disclosure can last even after the DNA binding domain-gene regulator becomes undetectable. The DBP fused to a repression domain can become undetectable after at least 3 days. In some embodiments, the DNA binding domain fused to a repression domain (e.g.. an RNBD-TF, a MAP- NBD-TF, or TALE-TF) can become undetectable after at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In some embodiments, qPCR or imaging via the FLAG-tag can be used to confirm that the DBP-TF is no longer detectable.C. Imaging Moieties

[0156] A DBP can be linked to a fluorophore, such as Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7, GFP, or mCHERRY. A DBP can be linked to a biotinylation reagent.Genes and Indications of Interest

[0157] In some embodiments, genome editing can be performed by fusing the DBP of the present disclosure to a nuclease, where the DBP binds for a particular genomic locus of interest. Genetic modification can involve introducing a functional gene for therapeutic purposes, knocking out a gene for therapeutic gene, or engineering a cell ex vivo (e.g., HSCs or CAR T cells) to be administered back into a subject in need thereof. For example, the genome editing complex can have a target site within a gene encoding PDCD1, CTLA4, LAG3, TET2, BTLA, HAVCR2, CCR5, CXCR4, TRA, TRB, B2M, albumin, HBB, HBA1, TTR, NR3C1, CD52, CBLB, TGFBR1, SERPINA1, CEP290, DMD, CFTR, IL2RG, CS-1, a erythroid specific enhancer of the BCL11A gene, or HBV genomic DNA in infected cells. In some embodiments, a genome editing complex can cleave double stranded DNA at a target site in order to insert a chimeric antigen receptor (CAR), alpha-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), or Factor 9 (F9). Cells, such as hematopoietic stem cells (HSCs) and T cells, can be engineered ex vivo with the genome editing complex. Alternatively, genome editing complexes can be directly administered to a subject in need thereof.

[0158] The subject receiving treatment can be suffering from a disease such as transthyretin amyloidosis (ATTR), HIV, glioblastoma multiforme, cancer, acute lymphoblastic leukemia, acute myeloid leukemia, beta-thalassemia, sickle cell disease, MPSI, MPSII, Hemophilia B, multiple myeloma, melanoma, sarcoma, Leber congenital amaurosis (LCA10), CD 19 malignancies, BCMA- related malignancies, duchenne muscular dystrophy (DMD), cystic fibrosis, alpha- 1 antitrypsin deficiency, X-linked severe combined immunodeficiency (X-SCID), or Hepatitis B.

[0159] In some embodiments, an RNBD of the present disclosure can be used to modify a target cell. The target cell can itself be unmodified or modified. For example, an unmodified cell can be edited with a DBP of the present disclosure to introduce an insertion, deletion, or mutation in itsgenome. In some embodiments, a modified cell already having a mutation can be repaired with an DBP of the present disclosure.

[0160] In some instances, a target cell is a cell comprising one or more single nucleotide polymorphism (SNP). In some instances, an DBP-nuclease described herein is designed to target and edit a target cell comprising a SNP.

[0161] In some cases, a target cell is a cell that does not contain a modification. For example, a target cell can comprise a genome without genetic defect (e.g., without genetic mutation) and an DBP-nuclease described herein can be used to introduce a modification (e.g., a mutation) within the genome.

[0162] A target cell may be obtained from a primate. The primate may be a human, or a nonhuman primate. The cell may be obtained from a cell sample may be obtained from a human. For example, the cell sample may comprise cells obtained from blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, pleural effusion, amniotic fluid, chorionic villus sample, vaginal fluid, interstitial fluid, buccal swab sample, sputum, bronchial lavage, Pap smear sample, or ocular fluid. The cell sample may comprise cells obtained from a blood sample, an aspirate sample, or a smear sample.

[0163] The cell sample may be a circulating tumor cell sample. A circulating tumor cell sample may comprise lymphoma cells, fetal cells, apoptotic cells, epithelia cells, endothelial cells, stem cells, progenitor cells, mesenchymal cells, osteoblast cells, osteocytes, hematopoietic stem cells (HSC) (e.g., a CD34+ HSC), foam cells, adipose cells, transcervical cells, circulating cardiocytes, circulating fibrocytes, circulating cancer stem cells, circulating myocytes, circulating cells from a kidney, circulating cells from a gastrointestinal tract, circulating cells from a lung, circulating cells from reproductive organs, circulating cells from a central nervous system, circulating hepatic cells, circulating cells from a spleen, circulating cells from a thymus, circulating cells from a thyroid, circulating cells from an endocrine gland, circulating cells from a parathyroid, circulating cells from a pituitary, circulating cells from an adrenal gland, circulating cells from islets of Langerhans, circulating cells from a pancreas, circulating cells from a hypothalamus, circulating cells from prostate tissues, circulating cells from breast tissues, circulating cells from circulating retinal cells, circulating ophthalmic cells, circulating auditory cells, circulating epidermal cells, circulating cells from the urinary tract, or combinations thereof.

[0164] The cell can be a T cell. For example, in some embodiments, the T cell can be an engineered T cell transduced to express a chimeric antigen receptor (CAR). The CAR T cell can be engineered to bind to BCMA, CD19, CD22, WT1, L1CAM, MUC16, R0R1, or LeY.

[0165] A cell sample may be a peripheral blood mononuclear cell sample.

[0166] A cell sample may comprise cancerous cells. The cancerous cells may form a cancer which may be a solid tumor or a hematologic malignancy. The cancerous cell sample may comprise cells obtained from a solid tumor. The solid tumor may include a sarcoma or a carcinoma.Exemplary sarcoma cell sample may include, but are not limited to, cell sample obtained from alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MEH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasms with perivascular epitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epitheioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, or telangiectatic osteosarcoma.

[0167] Exemplary carcinoma cell samples may include, but are not limited to, cell samples obtained from an anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0168] The cancerous cell sample may comprise cells obtained from a hematologic malignancy. Hematologic malignancy may comprise a leukemia, a lymphoma, a myeloma, a nonHodgkin’s lymphoma, or a Hodgkin’s lymphoma. The hematologic malignancy may be a T-cell based hematologic malignancy. The hematologic malignancy may be a B-cell based hematologic malignancy. Exemplary B-cell based hematologic malignancy may include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non- CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B- cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B- cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. Exemplary T-cell based hematologic malignancy may include, but are not limited to, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gammadelta T-cell lymphoma, lymphoblastic lymphoma, nasal NK / T-cell lymphomas, or treatment-related T-cell lymphomas.

[0169] A cell sample described herein may comprise a tumor cell line sample. Exemplary tumor cell line sample may include, but are not limited to, cell samples from tumor cell lines such as 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3 / Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC / 9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH / 2A, SNU-398, PLHC-1, HepG2 / SF, OCI-Lyl, OCLLy2, OCLLy3, OCLLy4, OCI-Ly6, OCI-Ly7, OCI-LylO, OCI-Lyl8, OCI-Lyl9, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.

[0170] A cell sample may comprise cells obtained from a biopsy sample, necropsy sample, or autopsy sample.

[0171] The cell samples (such as a biopsy sample) may be obtained from an individual by any suitable means of obtaining the sample using well-known and routine clinical methods.Procedures for obtaining tissue samples from an individual are well known. For example, procedures for drawing and processing tissue sample such as from a needle aspiration biopsy are well-known and may be employed to obtain a sample for use in the methods provided. Typically, for collection of such a tissue sample, a thin hollow needle is inserted into a mass such as a tumor mass for sampling of cells that, after being stained, will be examined under a microscope.

[0172] A cell may be a live cell. A cell may be a eukaryotic cell. A cell may be a yeast cell. A cell may be a plant cell. A cell may be obtained from an agricultural plant.EXAMPLES

[0173] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.EXAMPLE 1Truncation of LEGq and LEGm to engineer compact DNA-binding proteins

[0174] Schematics of full-length natural DNA-binding proteins, WP_058473422 from (SEQ ID NO:1) from L. quateirensis “LEGq” and WP_058451450 from L. maceachernii “LEGm”, are shown in Fig. 1 A. LEGq DNA-binding protein (DBP) includes a 143 amino acids long N-cap region, ten RUs, and a 162 amino acids long C-cap region. LEGm DNA-binding protein (DBP) includes a 99 amino acids long N-cap region, twelve RUs, and a 313 amino acids long C-cap region.

[0175] A 3X-FLAG tag and a SV40 NLS was added at the N-terminus of the LEGq and LEGm DBPs and an HA-tag was added at the C-terminus of the LEGq and LEGm DBPs enable protein retention during SELEX.

[0176] In vitro transcription / translation (IVTT) kits were used to produce the DBPs. The IVTT proteins were subjected to three SELEX cycles of: allowing proteins to bind to doublestranded DNA; immobilization of proteimDNA complexes on anti-HA magnetic beads and washing off unbound DNA; PCR amplification of bound DNA. In this way, the DNA molecules bound by the proteins were used as input for successive rounds of binding to the LEGq and LEGm DBPs,enriching the preferred binding sequence of these proteins within the pool of DNA molecules at each cycle.

[0177] PCR samples from each SELEX cycle were diluted 1:50 and amplified using indexed primers recognizing the common adapters flanking the 25-nt random sequences. PCRs were pooled, purified, and sequenced on an Illumina MiniSeq instrument.SELEX seq Data analysis

[0178] Sequence reads from the MiniSeq were de-multiplexed using the index sequences of the primers to assign sequence reads to individual SELEX samples. These reads were then filtered to require constant sequences matching the common adapters flanking a variable region of 25 nucleotides. A random sample of 5,000 reads from each sample was used as input to the GADEM motif finder (Li et al. 2009, Journal of Computational Biology, 16(2):317-29). Discovered motifs were visualized using the “ceqlogo” function of the MEME suite (Bailey et al. 2015, Nucleic Acids Research, 43(Wl):W39-49).

[0179] As shown in Fig. 1A, the LEGq DBP binds to the sequence G / AAGTGTCTCG (SEQ ID NO: 14). The LEGm DBP binds to the sequence GTCAGGACTCT (SEQ ID NO: 15).

[0180] Fig. 2 provides a schematic of various truncations designed for the N- and C-cap regions of the LEGq and LEGm DBPs. TEF plasmids with encoded LEG proteins with C-cap and N- cap truncations were prepared by Twist company. The DNA construct sequences were codon optimized according to Twist algorithm. Some of these DNA sequences failed Twist algorithm for feasibility of assembly or were not successfully produced.

[0181] Fig. 3 shows a Western blot for detecting the listed LEGm proteins. Fig. 4 shows a Western blot for detecting the listed LEGm and LEGq DBPs.

[0182] Figs. 5-18 provide the sequences of DNA bound by the listed LEGq DBPs. The results are summarized in Fig. 19.

[0183] These data show that the C-cap region can be truncated to a fragment 17 amino acid in length without significantly affecting the binding specificity of the DBPs. The N-cap region could be truncated to a fragment 67 amino acids in length without significantly affecting the binding specificity of the DBPs.

[0184] Figs. 20-29 provide the sequences of DNA bound by the listed LEGm DBPs. The results are summarized in Figs. 30 and 31. Fig. 30 provides colorimetric ELISA DNA-Protein binding assay. IVTT LEGm DBPs and biotinylated dsDNA oligo interact in binding buffer (dsDNA target oligo with binding sequence for LEGm proteins and negative control dsDNA oligo wereused). Biotinylated dsDNA binds to streptavidin on plate wells’ surface. After washes, mouse anti- FLAG antibody was added. After washes, HRP labeled anti-mouse antibody was added. Following washing to remove unbound HRP labeled anti-mouse antibody, the developing solution was added and absorbance was measured at 450 nm.

[0185] While N-cap truncations reduced biding to the consensus DNA sequence, C-cap truncations (e.g.C80) rescued the binding. A LEGm DBP with a N-cap fragment having 62 amino acids and a C-cap fragment having 80 amino acids retained binding specificity.

[0186] The SEQ ID NOs for the LEGq N-cap, RUs, and C-cap are below:

[0187] LEGq N-Cap fragments were designed as follows:

[0188] The SEQ ID NOs for the LEGq N-cap fragments are provided below:

[0189] LEGq C-Cap fragments were designed as follows:

[0190] The SEQ ID NOs for the LEGq C-cap fragments are provided below:

[0191] The SEQ ID NOs for the LEGm N-cap, RUs, and C-cap are below:

[0192] The LEGm N-cap fragments were designed as follows:

[0193] The SEQ ID NOs for the LEGm N-cap fragments are provided below:

[0194] The LEGm C-cap fragments were designed as follows:

[0195] The SEQ ID NOs for the LEGm C-cap fragments are provided below:

[0196] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the ait without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS1. An isolated polypeptide comprising: a N-cap region; a plurality of repeat units (RUs); and a C-cap region, wherein N-cap region is truncated relative to the full-length N-cap region of a Legionella quateirensis (L. qualeirensis) DNA-binding polypeptide, and / or wherein C-cap region is truncated relative to the full-length C-cap region of a Legionella quateirensis (L. quateirensis) DNA-binding polypeptide, wherein the RUs are arranged to bind to a target nucleic acid and wherein the isolated polypeptide binds to the target nucleic acid.

2. The isolated polypeptide of claim 1, wherein the N-cap region comprises a deletion of at least 24 amino acids at the N-terminus relative to the full-length N-cap region.

3. The polypeptide of claim 1 or 2, wherein the N-cap region is at least 24 amino acids long.

4. The polypeptide of any one of claims 1-3, wherein the N-cap region comprises a deletion of at least 51 amino acids at the N-terminus relative to the full-length N-cap region.

5. The polypeptide of any one of claims 1-4, wherein the N-cap region comprises a deletion of at least 76 amino acids at the N-terminus relative to the full-length N-cap region.

6. The polypeptide of any one of claims 1-5, wherein the N-cap region comprises a deletion of at least 100 amino acids at the N-terminus relative to the full-length N-cap region.

7. The polypeptide of any one of claims 1-6, wherein the N-cap region comprises a deletion of at least 119 amino acids at the N-terminus relative to the full-length N-cap region.

8. The polypeptide of any one of claims 1-7, wherein the N-cap region comprises a deletion of up to 119 amino acids at the N-terminus relative to the full-length N-cap region.

9. The polypeptide of any one of claims 1-8, wherein the C-cap region comprises a deletion of at least 26 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. quateirensis .

10. The polypeptide of any one of claims 1-9, wherein the C-cap region is at least 17 amino acids long.

11. The polypeptide of claim 1-10, wherein the C-cap region comprises a deletion of at least 49 amino acids at the C-terminus relative to the full-length C-cap region of the DNA binding protein of L. quateirensis.

12. The polypeptide of claim 1-11, wherein the C-cap region comprises a deletion of at least 80 amino acids at the C-terminus relative to the full-length C-cap region.

13. The polypeptide of claim 1-12, wherein the C-cap region comprises a deletion of at least 99 amino acids at the C-terminus relative to the full-length C-cap region.

14. The polypeptide of claim 1-13, wherein the C-cap region comprises a deletion of at least 132 amino acids at the C-terminus relative to the full-length C-cap region.

15. The polypeptide of claim 1-14, wherein the C-cap region comprises a deletion of at least 145 amino acids at the C-terminus relative to the full-length C-cap region.

16. The polypeptide of claim 1-14, wherein the C-cap region comprises a deletion of up to 145 amino acids at the C-terminus relative to the full-length C-cap region.

17. An isolated polypeptide comprising; a N-cap region; a plurality of repeat units; and a C-cap region,wherein N-cap region is truncated relative to the full-length N-cap region of a Legionella maceachernii (L. maceachemii) DNA-binding polypeptide, and / or wherein C-cap region is truncated relative to the full-length C-cap region of a Legionella maceachernii (L. maceachernii) DNA-binding polypeptide, and wherein the isolated polypeptide binds to DNA.

18. The isolated polypeptide of claim 17, wherein the N-cap region comprises a deletion of at least 14 amino acids at the N-terminus relative to the full-length N-cap region.

19. The isolated polypeptide of claim 17 or 18, wherein the N-cap region is at least 22 amino acids long.

20. The isolated polypeptide of any one of claims 17-19, wherein the N-cap region a deletion of at least 37 amino acids at the N-tcrminus relative to the full-length N-cap region.

21. The isolated polypeptide of any one of claims 17-20, wherein the N-cap region a deletion of at least 50 amino acids at the N-terminus relative to the full-length N-cap region.

22. The isolated polypeptide of any one of claims 17-21, wherein the N-cap region a deletion of at least 77 amino acids at the N-terminus relative to the full-length N-cap region.

23. The isolated polypeptide of any one of claims 17-22, wherein the N-cap region a deletion of up to 77 amino acids at the N-terminus relative to the full-length N-cap region.

24. The isolated polypeptide of any one of claims 17-23, wherein the C-cap region comprises a deletion of at least 44 amino acids at the C-terminus relative to the full-length C-cap region of the Legionella maceachemii DNA-binding polypeptide.

25. The isolated polypeptide of any one of claims 17-24, wherein the C-cap region comprises a deletion of at least 76 amino acids at the C-terminus relative to the full-length C-cap region.

26. The isolated polypeptide of any one of claims 17-25, wherein the C-cap region comprises a deletion of at least 106 amino acids at the C-terminus relative to the full-length C-cap region.

27. The isolated polypeptide of any one of claims 17-26, wherein the C-cap region comprises a deletion of at least 155 amino acids at the C-terminus relative to the full-length C-cap region.

28. The isolated polypeptide of any one of claims 17-27, wherein the C-cap region comprises a deletion of at least 184 amino acids at the C-terminus relative to the full-length C-cap region.

29. The isolated polypeptide of any one of claims 17-28, wherein the C-cap region comprises a deletion of at least 206 amino acids at the C-terminus relative to the full-length C-cap region.

30. The isolated polypeptide of any one of claims 17-29, wherein the C-cap region comprises a deletion of at least 233 amino acids at the C-terminus relative to the full-length C-cap region.

31. The isolated polypeptide of any one of claims 17-30, wherein the C-cap region comprises a deletion of at least 251 amino acids at the C-terminus relative to the full-length C-cap region32. The isolated polypeptide of any one of claims 17-31, wherein the C-cap region comprises a deletion of at least 283 amino acids at the C-terminus relative to the full-length C-cap region.

33. The isolated polypeptide of any one of claims 17-31, wherein the C-cap region comprises a deletion of up to 283 amino acids at the C-terminus relative to the full-length C-cap region.

34. The isolated polypeptide of any one of claims 17-33, wherein the C-cap region is at least 30 amino acids long.

35. The isolated polypeptide of any one of claims 1-34, wherein the isolated polypeptide is a fusion protein comprising a heterologous domain.

36. The isolated polypeptide of claim 35, wherein the heterologous domain is a transcriptional activator or a transcriptional repressor.

37. The isolated polypeptide of claim 35, wherein the heterologous domain is a nuclease domain.

38. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of claims 1-37.

39. The nucleic acid of claim 38, wherein the nucleotide sequence is operably linked to an expression control element.

40. A vector comprising the nucleic acid of claim 38 or 39.

41. The vector of claim 40, wherein the vector is a viral vector.

42. The vector of claim 41, wherein the viral vector is an adcno-associatcd viral vector or a Icntiviral vector.

43. A cell comprising: the polypeptide of any one of claims 1-37, the nucleic acid of claim 38 or 39, or the vector of any one of claims 40-42.

44. A composition comprising: the polypeptide of any one of claims 1-37, the nucleic acid of 38 or 39, or the vector of any one of claims 40-42, or the cell of claim 43; and a pharmaceutically acceptable carrier.

45. Use of a polypeptide of any one of claims 1-37, the nucleic acid of 38 or 39, or the vector of any one of claims 40-42, or the cell of claim 43, or a composition of claim 44 in a method of gene editing and / or modifying expression of a gene in a cell.

46. The use of claim 45, wherein the repeat units (RUs) bind to a target site in the genome of the cell.

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