Engineered high-efficiency compact nucleases and compositions, systems, and methods thereof

Abstract

The present disclosure provides enzymes, compositions, methods, and systems for nucleic acid editing. Particularly, the disclosure relates to engineered nucleases which are compact and highly efficient, fusion proteins of the engineered nucleases, systems including the engineered nucleases and / or fusion proteins, and methods of using thereof.

Description

COLUM-44474.601ENGINEERED HIGH-EFFICIENCY COMPACT NUCLEASES AND COMPOSITIONS,SYSTEMS, AND METHODS THEREOFFIELD

[0001] The present disclosure relates to enzymes, compositions, methods, and systems for nucleic acid editing. Particularly, the disclosure relates to engineered Casl2j (also referred to as Cas or CasPhi) nucleases which are compact and highly efficient, fusion proteins of the engineered nucleases, systems including the engineered nucleases and / or fusion proteins, and methods of using thereof.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Nos. 63 / 745,590, filed January 15, 2025, and 63 / 746,754, filed January 17, 2025, the contents of which are herein incorporated by reference in their entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled COLUM_44474_601_SequenceListing.xml (Size: 94,191 bytes: and Date of Creation: January 13, 2026) is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under EY024698 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] In bacteria and archaea, CRISPR / Cas systems provide immunity by incorporating fragments of invading phage, virus, and plasmid DNA into CRISPR loci and using corresponding CRISPR RNAs (“crRNAs”) to guide the degradation of homologous sequences. By engineering and harnessing the functions of these CRISPR / Cas systems over the past decade, programmable gene editing using components of these systems provides great promise for therapeutic treatments. However, current CRISPR-Cas nucleases face significant limitations, particularly regarding size and efficiency, in many therapeutic applications. While larger nucleases like SpCas9 offer high efficiency, they suffer from lower fidelity and are too large for single AAV delivery. Smaller nucleases that can fit in AAV vectors typically demonstrateCOLUM-44474.601limited efficiency compared to their larger counterparts. Thus, there is a continuing need to expand the available nucleases which are efficient, precise, and suitable for use in genetic engineering methods and therapeutic applications, particularly in eukaryotic cells and organisms. SUMMARY

[0006] Provided herein are engineered nucleases. In some embodiments, the engineered nucleases comprise less than 100% amino acid sequence identity with wild-type Casl2j (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1. In some embodiments, the engineered nucleases have increased efficiency as compared to wild-type Casl2j.

[0007] In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at positions 26, 147, 258, or a combination thereof, relative to SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at amino acid position 26, 147, 258, or a combination thereof. In some embodiments, the at least one amino acid substitution comprises substitutions of at least two positions selected from position 26, position 147, or position 258. In some embodiments, the at least one amino acid substitution comprises amino acid substitutions at positions 26, 147, and 258.

[0008] In some embodiments, the at least one amino acid substitution comprises substitution with a positively charged amino acid. In some embodiments, the at least one amino acid substitution comprises substitution with a lysine.

[0009] In some embodiments, the engineered nuclease comprises an L26K, N147K, and / or E258K amino acid substitution with reference to SEQ ID NO: 1.

[0010] In some embodiments, the engineered nuclease has an amino acid sequence with at least 75% identity to SEQ ID NO: 2. In some embodiments, the engineered nuclease has an amino acid sequence with at least 90% identity to SEQ ID NO: 2. In some embodiments, the engineered nuclease has an amino acid sequence of SEQ ID NO: 2.

[0011] In some embodiments, the engineered nuclease further comprises a localization sequence, a tag sequence, a protein transduction domain sequence, or a combination thereof.

[0012] Further provided herein are fusion proteins comprising an engineered nuclease as disclosed herein and an effector domain.

[0013] Also provided herein are systems or kits comprising an engineered nuclease as disclosed herein, or a nucleic acid encoding the engineered nuclease. In some embodiments, theCOLUM-44474.601systems or kits further comprise at least one gRNA complementary to at least a portion of a target nucleic acid sequence, or a nucleic acid encoding the at least one gRNA.

[0014] In some embodiments, the nucleic acid encoding the engineered nuclease comprises a messenger RNA or a vector.

[0015] In some embodiments, the at least one gRNA is encoded by a nucleic acid different from the nucleic acid encoding the engineered nuclease. In some embodiments, the at least one gRNA is encoded by a nucleic acid also encoding the nucleic acid encoding the engineered nuclease.

[0016] In some embodiments, the at least one gRNA is complexed with the engineered nuclease.

[0017] In some embodiments, the at least one gRNA is a non-naturally occurring gRNA.

[0018] In some embodiments, the system or kit further comprises a target nucleic acid sequence.

[0019] In some embodiments, the system is a cell-free system.

[0020] Further provided herein are compositions and cells comprising an engineered nuclease as described herein, or a nucleic acid encoding thereof, or a system as described herein and cells.

[0021] Additionally, methods for nucleic acid modification are provided. In some embodiments, the methods comprise contacting a target nucleic acid sequence with an engineered nuclease, a system, a nucleic acid, or a composition as described herein.

[0022] In some embodiments, the target nucleic acid sequence is in a cell. In some embodiments, contacting a target nucleic acid sequence comprises introducing the system into the cell. In some embodiments, introducing the system into the cell comprises administering the system to a subject. In some embodiments, the administering comprises in vivo administration.

[0023] Also provided are medicaments comprising an engineered nuclease and / or a fusion protein as disclosed herein, a nucleic acid encoding the engineered nuclease or fusion protein, a system as disclosed herein, or a composition thereof, for use in modifying a target nucleic acid sequence.

[0024] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.COLUM-44474.601BRIEF DESCRIPTION OF THE DR WINGS

[0025] FIG. 1 is a graph of formation of indels (insertions and deletions) in p.C147F SAG HEK293T cells for various Cas nucleases including Casl2j.

[0026] FIGS. 2A and 2B are graphs of the indel formation in p.C147F SAG HEK293T cells for single amino acid variants and multiple amino acid variants of wild-type Casl2j, respectively. v3.5 Casl2j corresponds to SEQ ID NO: 2.

[0027] FIG. 3 is a graph of the comparison of wild-type Casl2j and v3.5 Casl2j targeting in p.C147F SAG HEK293T cells.

[0028] FIGS. 4A-4D show percent indel formation across additional target sites, as indicated, for v3.5 Casl2j (CasPhiX). FIG. 4B is a summary of the sites shown in FIG. 4A. FIG. 4D is a summary of the sites shown in FIG. 4C.

[0029] FIGS. 5A and 5B show mismatch comparison for v3.5 Casl2j (CasPhiX) and wildtype Casl2j (WT CasPhi) in p.C147F HEK293T cells or wild-type HEK293T cells, respectively.

[0030] FIGS. 6A-6C show an allele- specific strategy targeting SAG p.C147F. FIG. 6A is a schematic demonstrating how an allele- specific strategy is possible using a Cas 12 nuclease that targets a T-rich PAM allowing for discrimination between WT and disease allele. FIG. 6B is a graph of editing efficiency and specificity of various Cas 12 nucleases in WT HEK293T cells and homozygous SAG p.C147F HEK293T cells (n=3). FIG. 6C is a schematic showing the relative size, domains, and PAMs of various CRISPR-Cas systems.

[0031] FIGS. 7A-7C show modification of Casl2j-8 to increase editing efficiency. FIG. 7A is a partial sequence-alignment of Casl2j-8 to other compact Cas nucleases, focusing on conserved positively charged amino acids in DNA- and RNA-binding domains (SEQ ID NOs: 35-70). FIG.7B shows the 60 different amino acid changes introduced into Casl2j-8 and compared to WT Casl2j-8 in SAG p.C147F homozygous HEK293T cells (n=3). FIG. 7C shows amino acid changes that improved editing efficiency and double and triple mutation variants (FIG. 7C, top). Casl2j-8 editing efficiency and were compared to single amino acid mutations (n=3, FIG. 7C, bottom). FIG. 7D is a graph of editing efficiency of Casl2j-8 v3.5 (L26K, N147K, E258K) compared to WT Casl2j-8 in WT and homozygous SAG p.C147F HEK293T cells (n=3).

[0032] FIGS. 8A-8C show mismatch discrimination comparison of Casl2j-8 and Casl2j-8 v3.5 targeting SAG p.C147F. FIG. 8A shows two different crRNA spacer options available forCOLUM-44474.601targeting SAG p.C147F (SEQ TD NOs: 33 and 34). crRNA 1 is SEQ ID NO: 3, cRNA 2 is SEQ ID NO: 22. FIGS. 8B and 8C show single mismatch discrimination assay of Casl2j-8 (FIG. 8B) and Casl2j-8 v3.5 (FIG, 8C) in WT and homozygous SAG p.C147F HEK293T cells using crRNA 2. Similar analysis on crRNA 1 is in FIGS. 5A and 5B. All possible mismatches were introduced in position 16 and 17 (SEQ ID NOs: 23-28, respectively) of the crRNA 2 (SEQ ID NO: 22) spacer (n=3).

[0033] FIGS. 9A-9D show use of Casl2j-8 v3.5 in a humanized SAG p.C147F mouse model using AAV. FIG. 9A is a diagram demonstrating the design of humanized SAG mice by replacing exon 7 mouse sequence with human. FIG. 9B is a diagram of subretinal injection of AAV8-Y773F encoding Casl2j-8 v3.5 and crRNA 2 with MM (T>G). CMV is a ubiquitous promoter, and Casl2j-8 v3.5 is linked to GFP via P2A to allow for visualization of successful transduction. FIG. 9C shows a comparison of qAF images of untransduced and transduced retinas, indicating successful expression of the AAV. FIG. 9D is a graph of editing efficiency of transduced GFP+ retinal cells from WT homozygous SAG mice and homozygous SAG p.C147F mice.

[0034] FIGS. 10A-10D show comparisons of Casl2j-83.5 to other nucleases at multiple different genomic loci. FIG. 10A is a comparison of editing efficiency of Casl2j-8, Casl2j-8 v3.5, Ascasl2f v5.1, CasMINI, hfCasl2Max, and AsCasl2a targeting 12 different genomic loci using the same spacer sequence (n=3). FIG. 10B is a comparison of editing efficiency of Casl2j-8, Casl2j-8 v3.5, and SpCas9 targeting 7 different genomic loci using the same spacer region (n=3). FIG. 10C is a box-and-whiskers plot comparing Casl2 nucleases across 12 genomic loci from FIG. 10A. FIG. 10D is a box-and-whiskers plot comparing Casl2j-8, Casl2j-8 v3.5 and SpCas9 across 7 genomic loci from FIG. 10B.

[0035] FIG. 11 is a graph of the allele-specificity of different Cas nucleases, including v3.5 Casl2j (CasPhiX), for targeting p.Cl 10R RHO.

[0036] FIG. 12 is diagram of RHO p.Cl 10R allele- specific AAV vector.DETAILED DESCRIPTION

[0037] The disclosed enzymes, compositions, methods, and systems include engineered nucleases useful for nucleic acid modification.

[0038] The disclosure provides engineered Casl2j (also known as and referred to as Cas or CasPhi) nucleases. Over fifty different single amino acid mutated Casl2j variants wereCOLUM-44474.601developed, with an exemplary triple amino acid mutated Casl2j variant showing significantly improved efficiency compared to the wild-type version. This modified nuclease is, on average, twice as efficient as the wild-type nuclease at inducing indels and subsequently ablating the target gene(s). The engineered Cas nuclease can be combined with its respective CRISPR-RNA (crRNA) scaffold and be programmed to target any sequence in the genome that has a nearby PAM sequence.

[0039] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0040] The terms “comprise(s),” “include(s).” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9. and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0042] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.COLUM-44474.601

[0043] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination, such as, but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan.

[0044] The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role to play in an organism. For the purpose of this disclosure, it may be considered that genes include regions that 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 regions.

[0045] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.10046] As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strengthCOLUM-44474.601of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tmof the formed hybrid. Hybridization methods involve the annealing of one nucleic acid to another, complementary nucleic acid, e.g., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and “anneal” or “hybridize” through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA, 46'. 453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA, 4(y. 461 (1960), have been followed by the refinement of this process into an essential tool of modem biology. For example, hybridization and washing conditions are now well known and exemplified in Sambrook et al., supra. The conditions of temperature and ionic strength determine the “stringency” of the hybridization.

[0047] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double- stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g.. Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc„ 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” asCOLUM-44474.601used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or doublestranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0048] Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(1): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0049] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0050] The terms “protein,” “peptide.” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in aCOLUM-44474.601protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, engineered, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0051] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the twenty-three amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader. Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine. Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D”COLUM-44474.601isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, orDF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide.

[0052] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site. The systems can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.

[0053] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g.. children).Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, guinea pigs, and the like. Examples of nonmammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0054] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which it is naturally associated in nature and as found in nature, and / or the nucleic acid molecule or the polypeptide is associated with at least one other component with which it is not naturally associated in nature and / or that there is one or more changes in nucleic acid or amino acid sequence as compared with such sequence as it is found in nature and / or that the nucleic acid or polypeptide sequence was generated de novo, e.g., not based on or derived from any naturally occurring sequence.

[0055] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid. to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.COLUM-44474.601

[0056] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Engineered Nucleases

[0057] Disclosed herein are engineered Casl2j nucleases. Further disclosed are nucleic acids and vectors comprising a sequence encoding the engineered nucleases. The engineered nucleases may confer desirable traits (e.g., increased stability, increased activity) not found in the wild-type versions of the proteins. In some embodiments, the engineered nucleases show increased activity or utility in modifying a target nucleic acid compared to a protein not having the disclosed modifications. In some embodiments, the engineered nuclease has increased efficiency as compared to wild-type Casl2j. In some embodiments, the engineered nuclease results in an increased nucleic acid cleavage (single-stranded cleavage and / or double-stranded cleavage) in a target nucleic acid as compared to wild-type Casl2j.

[0058] Provided herein are engineered nucleases having less than 100% amino acid sequence identity with wild-type Casl2j (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1.

[0059] In some embodiments, the at least one amino acid substitution comprises substitutions within a DNA binding domain or a crRNA binding domain. In some embodiments, the at least one amino acid substitution comprises substitutions in a DNA binding domain and a crRNA binding domain. In some embodiments, the at least one amino acid substitution comprises substitutions in the domains corresponding to those shown in FIG. 7A. In some embodiments, the at least one amino acid substitution comprises any of the substitutions shown in FIGS. 7A-7C.

[0060] In some embodiments, the at least one amino acid substitution is at amino acid position 26, 121, 147, 186, 258, or a combination thereof. For example, the at least one amino acid substitution may include single amino acid substitutions at position 26, position 121, position 147, position 186, or position 258, or any combination of amino acid substitutions at position 26, position 121, position 147, position 186, and position 258.COLUM-44474.601

[0061] In some embodiments, the at least one amino acid substitution is at amino acid position 26, 147, 258, or a combination thereof. For example, the at least one amino acid substitution may include single amino acid substitutions at position 26, position 147, or position 258, or combination of amino acid substitutions at position 26, position 147, or position 258. In some embodiments, the at least one amino acid substitution comprises substitutions of at least two positions selected from position 26, position 147, or position 258. In some embodiments, the at least one amino acid substitution comprises amino acid substitutions at positions 26, 147, and 258.

[0062] In some embodiments, the at least one amino acid substitution comprises substitution with a positively charged amino acid. In some embodiments, the at least one amino acid substitution comprises one or more substitutions of a negatively charged or neutral amino acid with a positively charged amino acid. In some embodiments, the at least one amino acid substitution comprises substitution with a lysine. In some embodiments, the at least one amino acid substitution comprises two or more substitutions of a negatively charged or neutral amino acid with a lysine.

[0063] In some embodiments, the engineered nuclease comprises an L26K, A121Q, N 147K, S186G, and / or E258K amino acid substitution with reference to SEQ ID NO: 1. In some embodiments, the engineered nuclease comprises an L26K, N147K, and / or E258K amino acid substitution with reference to SEQ ID NO: 1. In some embodiments, the engineered nuclease comprises an L26K, N147K, and E258K amino acid substitution with reference to SEQ ID NO: 1.

[0064] In some embodiments, the engineered nuclease has an amino acid sequence with at least 75% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 2. In some embodiments, the engineered nuclease has an amino acid sequence of SEQ ID NO: 2.

[0065] Any of the proteins described or referenced herein may comprise one or more additional amino acid substitutions, additions, and / or deletions. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe),COLUM-44474.601tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0066] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

[0067] In some embodiments, the engineered nucleases comprise one or more amino acid substitutions configured to fully or partially catalytically inactivate the nuclease. For example, the engineered nucleases may comprise one or more amino acids substitutions in one or more of the catalytic domains (e.g., a RuvC domain), resulting in full or partial inhibition of nuclease functionality. Full inhibition renders the engineered nuclease completely absent of any nuclease functionality.COLUM-44474.601

[0068] Any of the polypeptides (e.g., single polypeptides or fusion polypeptides) disclosed herein may further comprise one or more peptides fused to the polypeptide. The one or more peptides encompass both short amino acid sequences or protein or protein domain sequences. In some embodiments, the engineered nuclease further comprises a localization or signal sequence (e.g., nuclear localization sequence), a sequence tag (e.g., a tag for detection, purification, and / or monitoring expression), a protein transduction domain sequence, or a combination thereof. In some embodiments, the engineered nuclease is separated from the one or more peptides with a linker.

[0069] The engineered nucleases may comprise a nuclear localization sequence (NLS). The nuclear localization sequence may be appended to the N-terminus, the C-terminus, or embedded in the engineered nucleases (e.g., inserted internally within the open reading frame (ORF)). The engineered nucleases may comprise one or more nuclear localization sequences. The nuclear localization sequence may comprise any amino acid sequence known in the art to functionally tag or direct a protein for import into a cell’s nucleus (e.g.. for nuclear transport). Usually, a nuclear localization sequence comprises one or more positively charged amino acids, such as lysine and arginine.

[0070] In some embodiments, the NLS is a monopartite sequence. A monopartite NLS comprises a single cluster of positively charged or basic amino acids. In some embodiments, the monopartite NLS comprises a sequence of K-K / R-X-K / R, wherein X can be any amino acid. Exemplary monopartite NLSs include, without limitation, those from the SV40 large T-antigen, c-Myc, and TUS-proteins.

[0071] In some embodiments, the NLS is a bipartite sequence. Bipartite NLSs comprise two clusters of basic amino acids, separated by a spacer of about 9-12 amino acids. Exemplary bipartite NLSs include the NLS of nucleoplasmin, the NLS of EGL-13, the bipartite SV40 NLS.

[0072] The engineered nucleases may comprise an epitope or purification tag (e.g.. 3xFLAG tag, an HA tag, a Myc tag, a poly-histidine tag, and the like). In some embodiments, the epitope or purification tag may be adjacent, either upstream or downstream, to a nuclear localization sequence. The epitope tags may be at the N-terminus, a C-terminus, or a combination thereof of the corresponding engineered nuclease.COLUM-44474.601Fusion Proteins

[0073] The present disclosure also provides fusion proteins comprising the engineered nucleases described herein and one or more effector or functional domains. The fusion proteins are not limited by orientation or directionality of the engineered nuclease and the one or more effector domains. For example, any single effector domain may be fused to the N-terminus or C-terminus of the engineered nuclease, in any orientation, e.g., N-terminus to N-terminus, C-terminus to C-terminus, N-terminus to C-terminus, or C -terminus to N-terminus, and directly or indirectly (e.g., fused to another effector domain fused to the N-terminus).

[0074] Effector or functional domains are proteins or fragments thereof that can modify, regulate, or act as a tag for a target nucleic acid. For example, an effector domain can be used to target enzymatic activities to a nucleic acid sequence which the engineered nuclease targets (e.g., by way of a guide RNA, described elsewhere herein). In some embodiments, an effector domain is a fragment of protein that has been separated from its natural DNA binding domain and engineered to be part of a fusion protein with an engineered nuclease described herein. In some embodiments, an effector domain is a protein which normally binds to other proteins or factors for recruitment to a specific or non-specific nucleic acid.

[0075] The effector domain may comprise a number of functionalities, including but not limited to, nuclease function, recombinase function, epigenetic modifying function, transposase function, integrase function, resolvase function, invertase function, protease function, DNA methyltransferase function, DNA demethylase function, histone acetylase function, histone deacetylase function, transcriptional repressor function, transcriptional activator function, DNA binding protein function, transcription factor recruiting protein function, nuclear-localization signal function, DNA editing function (e.g., deaminase) or any combination thereof. For example, some effector domains function in transcriptional regulation via their ability to interact with the basal transcriptional machinery and general co-activators, interact with other transcription factors to allow cooperative binding, and / or directly or indirectly recruit histone and chromatin modifying enzymes.

[0076] For example, the at least one effector domain may comprise activator and / or repressor activity that can affect transcription upstream and downstream of coding regions, and can be used to activate or repress gene expression. In some embodiments, the at least one effector domain may include domains from transcription factors (activators, repressors, coactivators, co-COLUM-44474.601repressors), silencers, and / or chromatin associated proteins and their modifiers (e.g., methylases, demethylases, acetylases and deacetylases).

[0077] Accordingly, in some embodiments, a fusion protein as disclosed herein having a transcription activator effector domain can be used to directly increase gene expression. In some embodiments, a fusion protein as disclosed herein comprising a transcriptional protein recruiting domain, or active fragment thereof, can be used to recruit transcriptional activators or repressors to a specific nucleic acid sequence to localize activators and repressors to modulate gene expression in a targeted manner.

[0078] In some embodiments, the at least one effector domains comprise transcriptional repressor function. Transcription repressors prevent, partially or completely, the transcription of genes near to its target site. Exemplary transcriptional repressors include, but are not limited to, KRAB-domain containing proteins, SID, and Spl.

[0079] In some embodiments, the at least one effector domains comprise transcriptional activator function. Transcriptional activators can be generally defined as proteins, or domains thereof, that bind to specific sites on promoter DNA and bring about increased transcription of specific genes through interactions with other proteins. Exemplary transcriptional activators include, but are not limited to, VP64, p65, p53, c-Myb, GATA-1, EKLF, MyoD, E2F, dTCF, Tat, HSF1, RTA and SET7 / 9.

[0080] In some embodiments, the at least one effector domains comprise DNA methyltransferase or DNA methylase function. DNA methyltransferases (DNMT’s) are a family of DNA modifying proteins composed of different isomers (e.g., DNMT1, DNMT3A, and DNMT3B). Other exemplary DNA methyltransferases include Sssl methylase. Alul methylase. Haelll methylase, Hhal methylase, and Hpall methylase. Their main mechanism of action is addition of a methyl group to the fifth carbon of a cytosine residue (5mc) located adjacent to a guanine residue.

[0081] In some embodiments, the at least one effector domains comprise DNA demethylase function. DNA demethylation can be mediated by at least three enzyme families: (i) the ten-eleven translocation (TET) family, mediating the conversion of 5mC into 5hmC; (ii) the AID / APOBEC family, acting as mediators of 5mC or 5hmC deamination; and (iii) the BER (base excision repair) glycosylase family involved in DNA repair.COLUM-44474.601

[0082] Kinases, phosphatases, and other proteins that modify or regulate other polypeptides involved in gene regulation are also useful as effector domains. Such modifiers are often involved in switching on or off transcription mediated by, for example, hormones. Other useful domains for regulating gene expression can also be obtained from the gene products of oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members) and their associated factors and modifiers.

[0083] The at least one effector domains can be used to target enzymatic activity to locations containing the target nucleic acid sequence to which the gRNA is directed. For example, in some embodiments, effector domains having integrase or transposase activity can be used to promote integration of exogenous nucleic acid sequence into specific nucleic acid sequence regions and / or eliminate (knock-out) specific endogenous nucleic acid sequence.

[0084] Integrases allow for the insertion of nucleic acids, for example, into a host genome (mammalian, human, mouse, rat, monkey, frog, fish, plant (including crop plants and experimental plants like Arabidopsis), laboratory or biomedical cell lines or primary cell cultures, C. elegans, fly (Drosophila), etc.). Integrases are found in a retrovirus such as HIV (human immunodeficiency virus) and lambda integrase.

[0085] In some embodiments, the at least one effector domains comprise transposase functionality. Transposases are enzymes that bind to the end of a transposon and catalyze its movement by a cut and paste mechanism or a replicative transposition mechanism. Exemplary transpoases include, but are not limited to, Tel transposase, Mosl transposase, Tn5 transposase, and Mu transposase

[0086] In some embodiments, the at least one effector domains modify epigenetic signals and thereby modify gene regulation, for example by promoting histone acetylase and histone deacetylase activity. The term “epigenetic modifier,” as used herein, refers to a protein or catalytic domain thereof having enzymatic activity that results in the epigenetic modification of DNA, for example, chromosomal DNA. Epigenetic modifications include, but are not limited to, histone modifications including methylation and demethylation (e.g., mono-, di- and trimethylation), histone acetylation and deacetylation, as well as histone ubiquitylation, phosphorylation, and sumoylation.10087] Histone acetylation and deacetylation are the processes by which the lysine residues within the N-terminal tail protruding from the histone core of the nucleosome are acetylated andCOLUM-44474.601deacetylated as part of gene regulation. These reactions are typically catalyzed by enzymes with histone acetyltransferase (HAT) or histone deacetylase (HD AC) activity. Histone acetyltransferases include GNAT family proteins (e.g., Gcn5, Gcn5L, p300 / CREB-binding protein associated factor (PCAF), Elp3, HPA2 and HAT1) and MYST family proteins (e.g., Sas3, essential SAS-related acetyltransferase (Esal), Sas2, Tip60, MOF, MOZ, MORE, and HB01). Histone deacetylases fall into four classes. Class I includes HDACs 1. 2, 3, and 8. Class II is divided into two subgroups, Class IIA and Class IIB. Class IIA includes HDACs 4, 5, 7, and 9 while Class IIB includes HDACs 6 and 10. Class III contains the Sirtuins and Class IV contains only HDAC11. Classes of HD AC proteins are divided and grouped together based on the comparison to the sequence homologies of Rpd3, Hosl and Hos2 for Class I HDACs, HDA1 and Hos3 for the Class II HDACs and the sirtuins for Class III HDACs.

[0088] The site-specific methylation and demethylation of histone residues are catalyzed by methyltransferases and demethylases, respectively. Histone methylases transfer methyl groups to amino acids (e.g.. lysine and arginine) of histone proteins, ultimately affecting gene transcription. Methylases include SET1, MLL, SMYD3, G9a, GLP, EZH2, and SETDB1.Histone demethylases catalyze the removal of methyl marks from histones, an activity associated with transcriptional regulation and DNA damage repair. Demethylases include, for example, KDM1A, KDM1B, KDM2A, KDM2B, UTX.UTY, Jumonji C (JmJC) domain-containing demethylases, and GSK-J4.

[0089] In some embodiments, the at least one effector domains comprise nuclease activity (e.g., endo- or exo-nuclease activity). A nuclease is an agent that induces a break in a nucleic acid sequence, e.g., a single or a double strand break in a double-stranded DNA sequence.Nucleases include those which cut at or near a preselected or specific sequence and those which are not site specific. For example, nucleases include, but are not limited to, zincfinger nucleases (ZFN), homing endonucleases, meganucleases, restriction enzymes,TAL effector nucleases, Argonaute nucleases, CRISPR nucleases, comprising, for example, Cas9, Cpfl, Csml, CasX or CasY nucleases, micrococcal nuclease, staphylococcal nuclease, DNase I, T7 endonuclease, T5 exonuclease, or catalytically active fragments thereof.

[0090] In some embodiments, the at least one effector domains comprise invertase activity. Invertase activity can be used to alter genome structure by swapping the orientation of a DNA fragment.COLUM-44474.601

[0091] In some embodiments, the at least one effector domains comprise recombinase activity. A recombinase is a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g.. integrases). Examples of serine recombinases include, without limitation, Hin, Gin, Tn3 (also known as TnpR), 0-six, CinH, ParA, y5, Bxbl, (|)C31, TP901, TGI, <|>BT1, R4, (|)RV1, ^FCl, MR11, Al 18, U153, and gp29. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK.101, HK022, and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange.

[0092] In some embodiments, the at least one effector domains comprise resolvase activity. Resolvases are site-specific recombinases that function to excise (as a circle) a segment of DNA contained between two recombination sites (called res) and include, for example, Ruv C resolvase, Holiday junction resolvase Hjc ,Tn3 and yd resolvase.

[0093] In some embodiments, the at least one effector domains comprise a peptide or polypeptide sequence responsive to a ligand, such as a hormone receptor ligand binding domain, including, for example, the ligand binding domains of the estrogen receptor, the glucocorticosteroid receptor, and the like. Such effector domains can be used to act as “gene switches,” and be regulated by inducers, such as small molecule or protein ligands, specific for the ligand binding domain.

[0094] In some embodiments, the at least one effector domain comprises sequences or domains of polypeptides that mediate direct or indirect protein-protein interactions, including, for example, a leucine zipper domain, a STAT protein N terminal domain, and / or an FK506 binding protein.

[0095] In some embodiments, the at least one effector domains comprise DNA editing function (e.g., deaminase, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, polymerase activity (e.g., reverse transcriptase), ligase activity, helicase activity, photolyase activity or glycosylase activity).COLUM-44474.601

[0096] Tn some embodiments, the activity mediated by the at least one effector domains is a non-biological activity, such as a fluorescence activity (e.g., fluorescent proteins), luminescence activity (e.g.. a luminescent protein or enzyme which results in luminescence when interacting with a substrate (e.g., luciferase)), or binding activity, such as those mediated by maltose binding protein (“MBP”), glutathione S transferase (GST), hexahistidine, c-myc, and the FLAG epitope, for facilitating detection, purification, monitoring expression, and / or monitoring cellular and subcellular localization of the polypeptide to which the effector domain is appended. In such embodiments, the fusion proteins can also be used as a diagnostic reagent, for example, to detect mutations in gene sequences, to purify restriction fragments from a solution, or to visualize DNA fragments of a gel.

[0097] The effector domains described herein are illustrative and merely provide the skilled artisan with examples of effectors that can be used in combination with the engineered nucleases and methods described herein.

[0098] In some embodiments, the at least one effector domain comprises a transcription activator, a transcription repressor, a base editor, a polymerase (e.g., a reverse transcriptase) an epigenetic modifier, a chromosomal locus imaging agent (e.g., fluorescent protein or protein tag), or a combination thereof.Systems and Compositions

[0099] Disclosed herein are systems and compositions that comprise an engineered nuclease or fusion protein, as described herein, or one or more nucleic acids encoding the engineered nuclease or fusion protein. In some embodiments, the systems may further comprise a guide RNA (gRNA) or a nucleic acid encoding a gRNA. In some embodiments, the engineered nuclease is part of a ribonucleoprotein (RNP) complex with the gRNA.

[0100] The gRNA may be a crRNA, crRNA / tracrRNA (or single guide RNA, sgRNA). The terms “gRNA,” “guide RNA,” “crRNA,” and “CRISPR guide” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the binding specificity of the nucleases. A “guide sequence” (used interchangeably herein with “spacer,” “crRNA spacer,” or “spacer sequence”) refers to the sequence that hybridizes to (complementary to, partially or completely) a target nucleic acid sequence (e.g., the genome in a host cell) and therefore determines the sequence specificity of the gRNA. In some embodiments, the gRNA guide sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,COLUM-44474.60198%, 99%, or at least 100% complementary to a target nucleic acid. Tn some embodiments, the guide sequence has at least one mismatch with the target nucleic acid. In some embodiments, the guide sequence has one. two, three, four, five, or six mismatches to the target nucleic acid.

[0101] The gRNA or guide sequence may be any length. In some embodiments, the guide sequence is 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 nucleotides in length. gRNAs or sgRNA(s) used in the present disclosure can be between about 5 and 100 nucleotides long, or longer (e.g., 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 5960, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer).

[0102] To facilitate gRNA design, many computational tools have been developed (See Prykhozhij et al. (PLoS ONE, 10(3): (2015)); Zhu et al. (PLoS ONE, 9(9) (2014)); Xiao et al. (Bioinformatics. Jan 21 (2014)); Heigwer et al. (Nat Methods. 11(2): 122-123 (2014)). Methods and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available pre-designed gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish, C. elegans), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases.

[0103] In addition to the guide sequence, in some embodiments, the gRNA may also comprise a scaffold sequence (e.g., tracrRNA). In some embodiments, such a chimeric gRNA may be referred to as a single guide RNA (sgRNA). Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, and Ran, et al. Nature Protocols (2013) 8:2281-2308, incorporated herein by reference in their entireties. In some embodiments, the at least one gRNA is encoded in a CRISPR RNA (crRNA) array.

[0104] In some embodiments, the gRNA does not comprise a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA furtherCOLUM-44474.601comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence.

[0105] The gRNA may be a non-naturally occurring gRNA.

[0106] The system may further comprise a target nucleic acid. The terms “target sequence,” “target nucleic acid,” and “target site” (e.g., a “target genomic DNA sequence”) are used interchangeably herein to refer to a polynucleotide (nucleic acid, gene, chromosome, genome, etc.) to which a guide sequence (e.g., a synthetic guide RNA) is designed to have complementarity, wherein hybridization between the target sequence and a guide sequence promotes the formation of a complex with the engineered nuclease, provided sufficient conditions for binding exist. The target sequence and guide sequence need not exhibit complete complementarity, provided that there is sufficient complementarity to cause hybridization and promote formation of a complex with the engineered nuclease. A target sequence may comprise any polynucleotide, such as DNA or RNA. The target nucleic acid may be single stranded or double stranded. Suitable DNA / RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art.

[0107] The target sequence may or may not be flanked by a protospacer adjacent motif (PAM) sequence. In certain embodiments, a nucleic acid-guided nuclease can only cleave a target sequence if an appropriate PAM is present, see, for example Doudna et al., Science, 2014, 346(6213): 1258096, incorporated herein by reference. A PAM can be 5' or 3' of a target sequence. A PAM can be upstream or downstream of a target sequence. In one embodiment, the target sequence is immediately flanked on the 3' end by a PAM sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In certain embodiments, a PAM is between 2-6 nucleotides in length. In some embodiments, the PAM may comprise a sequence of TTTN.

[0108] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. There may be mismatches distal from the PAM.COLUM-44474.601

[0109] In some embodiments, the systems further comprise one or more additional genome engineering tools. For example, the systems may further comprise nucleases, such as zinc finger nucleases (ZFNs) and / or transcription activator like effector nucleases (TALENs); transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases.

[0110] The system may be a cell free system.

[0111] Also disclosed is a cell comprising the system described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell (e.g., a cell of a non-human primate or a human cell).

[0112] The compositions or systems may further comprise an excipient or carrier. Excipients and carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and / or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, corn starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0113] In some embodiments, the excipient or carrier is pharmaceutically acceptable.Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides;COLUM-44474.601di saccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0114] The carrier may include a delivery vehicle. Delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Exemplary delivery vehicles include, but are not limited to, microparticle compositions comprising a variety of polymers, liposomes or lipid nanoparticles, viral vectors, ribonucleoprotein (RNP) complexes, and the like.

[0115] Microparticles can include, but are not limited to, liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. In some cases, microparticle can include one or more of the following: a poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, pseudopoly amino acids), polyhydroxybutyrate-related copolymers, polyanhydrides, polymethylmethacrylate, polyethylene oxide), lecithin, lipids, and phospholipids, in any combination thereof.

[0116] In some embodiments, a liposome or lipid nanoparticle encapsulates the disclosed systems, nucleic acids, or proteins (e.g., engineered nucleases or fusion proteins thereof).Methods of making lipid compositions include, for example, lipid film hydration, optionally coupled with sonication or extrusion, solvent evaporation (e.g., ethanol injection, ether injection, or reverse phase evaporation), solvent-diffusion method, hot homogenization process, detergent removal methods, or combinations thereof. Any naturally occurring or synthetic vesicle forming lipid or combinations thereof can be used, including for example, di-aliphatic chain lipids, such as phospholipids; diglycerides; di-aliphatic glycolipids; single lipids such as sphingomyelin or glycosphingolipid; steroidal lipids; hydrophilic polymer derivatized lipids; or mixtures thereof. Liposome and lipid nanoparticle compositions of the disclosure may include one or more cationic and / or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethyleneglycol (PEG)-lipid conjugates, and / or sterols. In some embodiments, the lipid nanoparticle comprises a cationic lipid and / or ionizable lipid, a neutral or non-cationic lipid, and cholesterol.

[0117] The liposomes and lipid nanoparticles described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components include, without being limited thereto, fatty alcohols, fatty acids, and / or anyCOLUM-44474.601other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.

[0118] The liposome and lipid nanoparticle compositions of the disclosure can also be targeting compositions, e.g., contain one or more targeting moieties or biodistribution modifiers on the surface. A targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target and are generally known in the art, for example ligands such as folic acid, proteins, antibody or antibody fragments, and the like.

[0119] The phrase “pharmaceutically acceptable,” as used in connection with the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the composition (e.g., the nucleic acids, vectors, cells, proteins, or polypeptides) and does not negatively affect the subject to which the composition(s) are administered. Any of the compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions. Nucleic Acids and Delivery

[0120] The present disclosure also provides for nucleic acids encoding an engineered nuclease, fusion protein, or system, as disclosed herein, and vectors containing or encoding these nucleic acids. The vectors may be used to propagate the nucleic acid in an appropriate cell and / or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0121] In certain embodiments, the nucleic acids are engineered for codon-optimization. It will be appreciated altering codons to those most frequently used in the cells or subject of interest allows for maximum expression. Such modified nucleic acid sequences are commonly described in the art as “codon-optimized.” In some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g., about 65%, about 70%, about 75%, aboutCOLUM-44474.60180%, about 85%, about 90%, about 95%, or about 98%) of the codons encoded therein are preferred codons to the subject of interest.

[0122] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode one or more of the engineered nuclease or components of the present systems. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell.

[0123] The vectors of the present disclosure may be delivered to a eukaryotic cell in a subject. Modification of the eukaryotic cells via the present system can take place in a cell culture, where the method comprises isolating the eukaryotic cell from a subject prior to the modification. In some embodiments, the method further comprises returning said eukaryotic cell and / or cells derived therefrom to the subject.

[0124] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding the disclosed engineered nucleases or components of the present system into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding the disclosed engineered nucleases or components of the present system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), and nucleic acids complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

[0125] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the necessary components of the system may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration. Drug selection strategies may be adopted for positively selecting for cells. A donor nucleic acid may contain one or more drug- selectable markers within the cargo. Then presuming that the original donor plasmid is removed, drug selection may be used to enrich for integrated clones. Colony screenings may be used to isolate clonal events.

[0126] A variety of viral constructs may be used to deliver the disclosed engineered nucleases or components of the present system to the targeted cells and / or a subject. NonlimitingCOLUM-44474.601examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al„ 2001 Nat. Medic. 7(l):33-40: and Walther W. and Stein U., 2000 Drugs. 60(2): 249-71, incorporated herein by reference.

[0127] In one embodiment, a nucleic acid encoding the disclosed engineered nucleases, fusion proteins, or components of the present system is contained in a plasmid vector that allows expression of the disclosed engineered nucleases or components of the present system and subsequent isolation and purification of from the recombinant vector. Accordingly, the disclosed engineered nucleases, fusion protein, or components of the present system disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[0128] To construct cells that express the disclosed engineered nucleases or components of the present system, expression vectors for stable or transient expression of the disclosed engineered nucleases or components of the present system may be constructed via conventional methods as described herein and introduced into host cells. For example, nucleic acids encoding the components of the disclosed engineered nucleases or components of the present system may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors / plasmids / viral vectors should be suitable for integration and replication in eukaryotic cells.

[0129] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms. The system may be used with various bacterial hosts.

[0130] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840. incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated hereinCOLUM-44474.601by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds.. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0131] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissuespecific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit betaglobin splice acceptor), TRE (Tetracycline response element promoter). Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-alpha (EFl-a) promoter with or without the EFl-a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0132] Moreover, inducible and tissue specific expression of a RNA, transmembrane proteins, or other proteins can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence.COLUM-44474.601Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various commercially available ubiquitous as well as tissue-specific promoters and tumor- specific are available, for example from InvivoGen. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto.

[0133] The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0134] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly-expressed genes like a-globin or P-globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcriptionCOLUM-44474.601of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0135] When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0136] The disclosed engineered nucleases or components of the present system may be delivered by any suitable means. In certain embodiments, the engineered nucleases or system is delivered in vivo. In other embodiments, the engineered nucleases or system is delivered to isolated / cultured cells (e.g., autologous iPS cells) in vitro to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition.

[0137] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0138] Any of the vectors comprising a nucleic acid sequence that encodes the disclosed engineered nucleases or components of the present system are also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA orCOLUM-44474.601RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding the disclosed polypeptides or components of the present system is a DNA molecule. In some embodiments, the nucleic acid encoding the disclosed polypeptides or components of the present system is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the disclosed polypeptides or components of the present system is an RNA molecule, which may be electroporated to cells.

[0139] Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1; 459( l-2):70-83). incorporated herein by reference.Methods of Use10140] The disclosure also provides methods of modifying a target nucleic acid sequence. The phrase “modifying a nucleic acid sequence” or “nucleic acid modification” as used herein, refers to modifying at least one physical feature of a nucleic acid sequence of interest. Nucleic acid modifications include, for example, single or double strand breaks, deletion, or insertion of one or more nucleotides, and other modifications that affect the structural integrity or nucleotide sequence of the nucleic acid sequence.

[0141] In some embodiments, the methods introduce a single strand or double strand break in the target nucleic acid sequence. In this respect, the disclosed systems may direct cleavage of one or two strands of a target nucleic acid sequence, such as within a target genomic DNA sequence and / or within the complement of the target sequence.COLUM-44474.601

[0142] In some embodiments, altering a nucleic acid sequence comprises a deletion. The deletion may be upstream or downstream of a nuclease binding site, so called unidirectional deletions. The deletion may encompass sequences on either side of the binding site, a bidirectional deletion. The deletion of the nucleic acid sequence may be of any size. The methods can be used to delete nucleic acids from a target sequence in a host cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence.

[0143] The methods may result in modifying the nucleic acid sequence as a result of one of more of the effector or functional domains as described above, rather than the nuclease function. For example, the methods may modulate the transcription of a target nucleic acid, may add or remove moieties from the target nucleic acid (e.g., methyl groups), may edit bases in the target nucleic acid (e.g., deaminate, depurinate, depyrimidinate), may unwind, replication, of combine target nucleic acids, and / or may add or remove moieties from histones (e.g., methylate, demethylate, acetylate, deacetylate, ubiquitinate, phosphorylate, sumoylate) bound to the nucleic acid.

[0144] The methods comprise contacting a target nucleic acid sequence with an engineered nuclease, composition, or system as described herein. In some embodiments, contacting a target nucleic acid sequence comprises introducing the engineered nuclease, composition, or system into a cell. As described above, the engineered nuclease, composition, or system, or nucleic acids encoding thereof, may be introduced into eukaryotic or prokaryotic cells by methods known in the art. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0145] In some embodiments, the target nucleic acid is a nucleic acid endogenous to a target cell. In some embodiments, the target nucleic acid is a genomic DNA sequence. The term “genomic,” as used herein, refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell. In some embodiments, the target nucleic acid encodes a gene or gene product. The term “gene product,” as used herein, refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, micro RNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA). In some embodiments, the target nucleic acid sequence encodes a protein or polypeptide.COLUM-44474.601

[0146] Polynucleotides containing the target nucleic acid sequence may include, but is not limited to, purified chromosomal DNA, total cDNA, cDNA fractionated according to tissue or expression state (e.g„ after heat shock or after cytokine treatment other treatment) or expression time (after any such treatment) or developmental stage, plasmid, cosmid, BAC, YAC, phage library, etc. Polynucleotides containing the target site may include DNA from organisms such as Homo sapiens. Mus domesticus, Mus spretus. Canis domesticus. Bos. Caenorhabditis elegans, Plasmodium falciparum, Plasmodium vivax, Onchocerca volvulus, Brugia malayi, Dirofilaria immitis, Leishmania, Zea maize, Arabidopsis thaliana, Glycine max, Drosophila melanogaster. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium tuberculosis, Aquifex, Thermus aquaticus, Pyrococcus furiosus, Thermus littoralis, Methanobacterium thermoautotrophicum, Sulfolobus caldoaceticus, and others.

[0147] The target nucleic acid may be single stranded or double stranded.

[0148] The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells, an effective amount of the described engineered nuclease, composition, or system, or nucleic acid(s) encoding thereof. In some embodiments, the engineered nuclease, composition, or system, or nucleic acid(s) encoding thereof is delivered to the tissue of interest by, for example, an intramuscular, intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods.

[0149] The engineered nuclease, composition, or system, or nucleic acid(s) encoding thereof, or ex vivo treated cells may be administered with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the engineered nuclease, composition, or system, or nucleic acid(s) encoding thereof, may be mixed, individually or in any combination, with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure.

[0150] In some embodiments, an effective amount of the engineered nuclease, composition, or system, or nucleic acid(s) encoding thereof, as described herein can be administered. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the termCOLUM-44474.601“effective amount” refers to that quantity of the components of the system such that successful DNA integration is achieved.

[0151] When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.

[0152] In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease.

[0153] The phrase “pharmaceutically acceptable,” as used in connection with the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and / or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[0154] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serumCOLUM-44474.601albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0155] The methods may be used for a variety of purposes. For example, the methods may include, but are not limited to, inactivation of a gene, methods of treating a subject suffering from a disease or disorder, and methods of treating a diseased cell.

[0156] The disclosed methods may modify a target DNA sequence in a cell so as to modulate expression of the target DNA sequence, e.g., expression of the target DNA sequence is increased, decreased, or completely eliminated (e.g., via deletion of a gene). The modifications of the target sequence may lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, gene knockdown, etc.

[0157] In another embodiment, the method of modifying a target sequence can be used to delete nucleic acids from a target sequence in a host cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule. Deletion of a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research.

[0158] In some embodiments, the methods described herein may be used to genetically modify a plant or plant cell. As used herein, genetically modified plants include a plant into which has been introduced an exogenous polynucleotide. Genetically modified plants also include a plant that has been genetically manipulated such that endogenous nucleotides have been altered to include a mutation, such as a deletion, an insertion, a transition, a transversion, or a combination thereof. For instance, an endogenous coding region could be deleted. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified plant is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region. The genetically modified plant may promote a desired phenotypic or genotypic plant trait.COLUM-44474.601

[0159] Genetically modified plants can potentially have improved crop yields, enhanced nutritional value, and increased shelf life. They can also be resistant to unfavorable environmental conditions, insects, and pesticides. The present systems and methods have broad applications in gene discovery and validation, mutational and cisgenic breeding, and hybrid breeding. The present methods may facilitate the production of a new generation of genetically modified crops with various improved agronomic traits such as herbicide resistance, herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, disease (e.g. bacterial, fungal, and viral) resistance, high yield, and superior quality. The present methods may also facilitate the production of a new generation of genetically modified crops with optimized fragrance, nutritional value, shelf-life, pigmentations (e.g., lycopene content), starch content (e.g., low-gluten wheat), toxin levels, propagation and / or breeding and growth time. See, for example, CRISPR / Cas Genome Editing and Precision Plant Breeding in Agriculture (Chen et al., Annu Rev Plant Biol. 2019 Apr 29;70:667-69), incorporated herein by reference.

[0160] The present method may confer one or more of the following traits to the plant cell: herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, resistance to fungal disease, and resistance to viral disease.

[0161] The present disclosure provides for a modified plant cell produced by the present method, a plant comprising the plant cell, and a seed, fruit, plant part, or propagation material of the plant. Transformed or genetically modified plant cells of the present disclosure may be as populations of cells, or as a tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, tuber, grain, animal feed, a field of plants, and the like. The present disclosure provides a transgenic plant. The transgenic plant may be homozygous or heterozygous for the genetic modification. Also provided by the present disclosure are transformed or genetically modified plant cells, tissues, plants, and products that contain the transformed or genetically modified plant cells. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants.COLUM-44474.601

[0162] The present system and method may be used to modify a plant stem cell. The present disclosure further provides progeny of a genetically modified cell, where the progeny can comprise the same genetic modification as the genetically modified cell from which it was derived. The present disclosure further provides a composition comprising a genetically modified cell.

[0163] In one embodiment, the transformed or genetically modified cells, and tissues and products comprise a nucleic acid integrated into the genome, and production by plant cells of a gene product due to the transformation or genetic modification.

[0164] Methods of introducing exogenous nucleic acids into plant cells are well known in the art. Such plant cells are considered “transformed.” DNA constructs can be introduced into plant cells by various methods, including, but not limited to PEG- or electroporation-mediated protoplast transformation, tissue culture or plant tissue transformation by biolistic bombardment, or the Agrobacterium-mediated transient and stable transformation. The transformation can be transient or stable transformation. Suitable methods also include viral infection (such as double stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whiskers technology, Agrobacterium-mediated transformation, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). Transformation methods based upon the soil bacterium Agrobacterium turnefaciens are useful for introducing an exogenous nucleic acid molecule into a vascular plant. The wild-type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred. An Agrobacterium-based vector is a modified form of a Ti plasmid, in which the tumor inducing functions are replaced by the nucleic acid sequence of interest to be introduced into the plant host.

[0165] Agrobacterium-mediated transformation generally employs cointegrate vectors or binary vector systems, in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences. A variety ofCOLUM-44474.601binary vectors are well known in the art and are commercially available, for example, from Clontech (Palo Alto, Calif.). Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the art. See., e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.: CRC Press (1993), incorporated herein by reference.

[0166] Microprojectile-mediated transformation also can be used to produce a transgenic plant. This method, first described by Klein et al. (Nature 327:70-73 (1987), incorporated herein by reference), relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine, or polyethylene glycol. The microprojectile particles are accelerated at high speed into an angiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad; Hercules Calif.).

[0167] In one embodiment, the present methods may be adapted to use in plants. The vectors may be optimized for transient expression of the present system in plant protoplasts, or for stable integration and expression in intact plants via the Agrobacterium-mediated transformation.

[0168] In certain embodiments, the present methods use a monocot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a monocot plant. In certain embodiments, the present methods use a dicot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a dicot plant.

[0169] The present methods may be used with various microbial species, including human pathogens that are medically important, and bacterial pests that are key targets within the agricultural industry, as well as antibiotic resistant versions thereof. The method may be designed to target any gene or any set of genes, such as virulence or metabolic genes, for clinical and industrial applications in other embodiments. For example, the present methods may be used to target and eliminate virulence genes from the population, to perform in situ gene knockouts, or to stably introduce new genetic elements to the metagenomic pool of a microbiome. The present systems and methods may be used to treat a multi-drug resistance bacterial infection in a subject. The present systems and methods may be used for genomic engineering within complex bacterial consortia.

[0170] The present systems and methods may be used to inactivate microbial genes. In some embodiments, the gene is an antibiotic resistance gene. For example, the coding sequence ofCOLUM-44474.601bacterial resistance genes may be disrupted in vivo by insertion of a DNA sequence, leading to non-selective re- sensitization to drug treatment.

[0171] The methods described here also provide for treating a disease or condition in a subject. The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells (e.g., disclosed T cells), a therapeutically effective amount of an engineered nuclease, composition, or system, as described herein. In some embodiments, the methods are used to treat a pathogen or parasite on or in a subject by altering the pathogen or parasite.

[0172] In some embodiments, the methods target a “disease-associated” gene. The term “disease-associated gene,” refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. A disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and / or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. Examples of genes responsible for such “single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, a-1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), P-hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin- specific peptidase 9Y, Y-linked (USP9Y). Other single gene or monogenic diseases are known in the art and described in, e.g., Chial. H. Rare Genetic Disorders: Learning About Genetic Disease Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1): 192 (2008); Online Mendelian Inheritance in Man (OMIM); and the Human Gene Mutation Database (HGMD). In another embodiment, the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (i.e., Mendelian) inheritance patterns are referred to in the art as a “multifactorial” or “polygenic” disease. Examples of multifactorial orCOLUM-44474.601polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip / palate, congenital heart defects, and neural tube defects. In another embodiment, the target DNA sequence can comprise a cancer oncogene. The present disclosure provides for gene editing methods that can ablate a disease-associated gene (e.g.. a cancer oncogene), which in turn can be used for in vivo gene therapy for patients. In some embodiments, the gene editing methods include donor nucleic acids comprising therapeutic genes.

[0173] In some embodiments, the disease or disorder is an ocular disease or disorder. In some embodiments, the disease or disorder is a retinal disease or disorder. In some embodiments, the disease or disorder is an inherited retinal disorder (IRD). In some embodiments, the disease or disorder comprises disease or disorder comprises retinal degeneration, retinitis pigmentosa, night blindness, macular dystrophy, vitelliform macular dystrophy (e.g., adult-onset vitelliform dystrophy (AVMD), Best vitelliform macular dystrophy (B VMD)), autosomal dominant vitreoretinochoroidopathy (ADVIRC), Leber congenital amaurosis, central areolar choroidal dystrophy, inherited macular degeneration, (e.g., macular dystrophy malattia leventinese / Doyne honeycomb retinal dystrophy) cone-rod dystrophy, bestrophinopathies, or a combination thereof.

[0174] In some embodiments, the disease or disorder is caused or mitigated by a disease-associated allele (e.g., autosomal dominant disease-associated allele) of: arrestin- 1 (SAG), rhodopsin (RHO), inosine 5 '-monophosphate dehydrogenase type I (IMPDH1), retinol isomerase retinal pigment epithelium (RPE) 65 (RPE65), epidermal growth factor-containing, fibulin-like extracellular matrix protein 1 (EFEMP1, also called fibulin-3), or bestrophin-1 (BEST1). Thus, in some embodiments, the target nucleic acid is arrestin- 1 (SAG), rhodopsin (RHO), inosine 5'-monophosphate dehydrogenase type I (IMPDH1), retinol isomerase retinal pigment epithelium (RPE) 65 (RPE65), epidermal growth factor-containing, fibulin-like extracellular matrix protein 1 (EFEMP1, also called fibulin-3), or bestrophin-1 (BEST1), or a disease-associated allele thereof.

[0175] Further, disclosed herein are methods to modify: arrestin- 1 (SAG), rhodopsin (RHO), inosine 5 '-monophosphate dehydrogenase type I (IMPDH1), retinol isomerase retinal pigment epithelium (RPE) 65 (RPE65), epidermal growth factor-containing, fibulin-like extracellularCOLUM-44474.601matrix protein 1 (EFEMP1, also called fibulin-3), or bestrophin-1 (BEST1), or a disease-associated allele thereof.

[0176] In some embodiments, the disease-associated allele is a C147F disease-associated allele of SAG. In some embodiments, the disease-associated allele is a C110R, R135W, T17M, D190N, R135L, N15S, G106R, T58R, G89D, and / or G90D disease-associated allele of RHO. In some embodiments, the disease-associated allele is a D226N or R190W disease-associated allele of IMPDH1. In some embodiments, the disease-associated allele is a D477G disease-associated allele of RPE65. In some embodiments, the disease-associated allele is a D226N or R345W disease-associated allele of EFEMP1. In some embodiments, the disease-associated allele is a D301E, R218H, or R218C disease-associated allele of BEST1.

[0177] In some embodiments, the system is configured to modify the disease-associated allele while not modifying a wild-type or non-pathogenic allele. Thus, the system may target the disease-associated allele based on the appropriate guide RNA sequence. In some embodiments, the methods further comprise determining the presence of the disease-associated allele.

[0178] In some embodiments, the systems or components thereof are configured for delivery to retinal cells. In some embodiments, the system is configured for delivery to rod and cone photoreceptor cells. For example, in some embodiments, the nucleic acids encoding the components may comprise a retinal cell (e.g., rod and / or cone photoreceptor cell) promoter which directs expression of the components in the retinal cells. Suitable retinal, rod, and / or cone photoreceptor cell promoters include, but are not limited to: 770En_454P(hG / ?M<5), a human GRM6 gene-derived, short promoter; promoters based on the 2.1 -kb human L-opsin promoter (pR2.1); promoter derived from the rhodopsin kinase (RK) gene; promoter derived from the rhodopsin gene; a promoter derived from the Nrl gene; murine rhodopsin promoter (mOP); G-protein-coupled receptor protein kinase 1 (GRK1) promoter; retinol-binding protein 3, interstitial (RBP3) promoter; RPE65 promoter; human inter-photoreceptor retinoid binding protein / retinol-binding protein 3 (IRBP) promoter; and retinaldehyde binding protein 1 (RLBP1 ) promoter. Additionally, or alternatively, the systems or components are configured for administration to the eye and / or retina, rather than systemic administration.

[0179] Administration may be through any suitable mode of administration, including but not limited to: intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular,COLUM-44474.601intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, topical, and inhalation. In some embodiments, the systems or components are delivered to the tissue(s) of interest. Such delivery may be either via a single dose, or multiple doses.

[0180] In some embodiments, an effective amount of the components of the systems, methods or compositions as described can be administered. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “effective amount” refers to that quantity of the components of the system such that successful modification of the disease-associated allele or successful modulation of expression of the disease-associated allele is achieved.

[0181] When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.Kits

[0182] Also within the scope of the present disclosure are kits that include the engineered nucleases, fusion proteins, nucleic acids, cells, compositions, systems, or components thereof as disclosed herein.

[0183] The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of administration to a subject to achieve the intended effect. The instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.

[0184] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. A kit may have a sterile accessCOLUM-44474.601port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0185] The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject.

[0186] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0187] The kit may further comprise a device for holding or administering the present system, polypeptides, or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and / or a syringe.

[0188] The present disclosure also provides for kits for performing nucleic acid modification and integration in vitro. Optional components of the kit include one or more of the following: buffer constituents, control plasmid, sequencing primers, cells.ExamplesMaterials and Methods

[0189] Plasmid constructions All CRISPR-Cas nuclease fragments and their respective sgRNAs were cloned into a mammalian SauriCas9 AAV-backbone expression vector (Addgene no. 135965). Expression of the nucleases were driven by a CMV promoter, and U6 was used to drive sgRNA expression. DNA fragments for plasmid construction were PCR amplified using SuperFi II (Thermofisher, 12361050) and assembled using In-Fusion Cloning (Takara Bio, 639650). sgRNA fragments were inserted in between two BbsI or Bsal restriction enzyme sites. Casl2j variants were generated by site-directed mutagenesis.

[0190] Cell culture HEK293T cells were cultured in DMEM supplemented with 10% (v / v) fetal bovine serum, 1% GlutaMax (Thermofisher, 35050061), and 1% non-essential amino acids (Thermofisher, 11140050). Cells were grown at 37°C with 5% CO2.

[0191] Site-directed Mutagenesis Site-directed mutagenesis was performed using the platnium superFi II DNA polymerase in accordance with the manufacturer’s instructions.COLUM-44474.601Briefly, primers were designed to make the desired amino acid change and PCR was then run using the WT Casl2j-8 nuclease with the desired primers. Dpnl digestion was then used to remove the original plasmid and then introduced into competent cells using heat shock. Cells were then cultured overnight in 37C on ampicillin-resistant plates. Colonies were picked the next day, expanded, and prepared for whole-plasmid sequencing.

[0192] In vitro evaluation ofindels Transfections were done in 24-well plates. 1 pg plasmid encoding the respective Cas nuclease and sgRNA were transfected using 1 pL lipofectamine 2000 (Thermofisher, 11668019) in 50 pL Opti-MEM (Thermofisher, 31985062). Two days after transfection 2 pg / mL puromycin (Thermofisher, Al 113803) was added to the media. Three days after puromycin selection cells were collected, genomic DNA (gDNA) was isolated, and the targeted region was amplified using site- specific primers with partial Illumina adapter sequences (Forward: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ (SEQ ID NO: 29), Reverse: 5’-GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’(SEQ ID NO: 30)) using SuperFi II, with 1 pL cell lysate used as templates. Amplicons were subsequently purified using the QIAquick PCR purification kit (Qiagen 28106), and subsequently sent for Illumina-based sequencing by Genewiz (Amplicon-EZ). Next-generation sequencing reads were generated for each sample using 2 x 250-bp paired-end reads. CRISPResso and Cas- Analyzer were used to analyze the data.

[0193] Establishing a homozygous SAGC147FHEK293T cell lines Transfections were conducted using PEMax (Addgene no. 174820), a pegRNA-evopreQl expressing plasmid (Addgene no. 174038), and a nsgRNA plasmid in combination with lipofectamine 2000 (Invitrogen 11668027). Three days post-transfection the cells were seeded in a 96-well plate at a concentration of 1 cell / well. Cells were given time to grow to confluency, at which point the cells were passaged, with some gDNA being collected from each well as previously described and amplified for genotyping. Genotyping was confirmed by Sanger sequencing by Genewiz.

[0194] Generation of humanized SAGWTand SAGC147Fmice A mixture of 0.15 pM SpCas9 sgRNAs (Integrated DNA Technologies (IDT), 5’-AGGGACAGATATCTACCTAT-3’ (SEQ ID NO: 31), 5’-CAAGATCCCCAAGAAGTAAG-3’(SEQ ID NO: 32), represented as DNA), 0.125 uM .S' Cas9 protein (IDT), and 5 ng / uL single- stranded oligodeoxynucleotides donor template (IDT, Megamer single-stranded DNA fragment) was injected into the pronuclei and cytoplasm of B6CBAF1 Fl zygotes. Healthy zygotes were transferred into the oviducts of pseudopregnantCOLUM-44474.601B6CBA Fl females. Resulting successful FO mice were then backcrossed into the C57BL / 6J background for five generations before being crossed to generate homozygous hSAGWT / hSAGWTand hSAGCI47F / hSAGCI47Fmice.

[0195] Genotyping humanized SAGW1and SAG71477mice Mouse tail pieces were cut and gDNA was collected using the DNeasy Blood and Tissue kit (Qiagen 69506) and amplified using primers flanking the humanized SAG region. The PCR product was then incubated with BamHI (New England Biolabs, R0136S) at 37° C overnight. The resulting product was then analyzed by agarose gel electrophoresis.

[0196] Designing AAV vectors The existing CMV-Casl2j-P2A-Puro with the U6-crRNA2 17-2 was modified by replacing the puromycin cassette with GFP using infusion cloning. The resulting CMV-Casl2j-P2A-GFP with U6-crRNA2 17-2 plasmid was then packaged into the AAV8 capsid with the Y773F modification by Packgene.

[0197] Delivering AAVs to the retina Mice aged four to six weeks were anesthetized via intraperitoneal administration of a ketamine (10 mg / mL) and xylazine (1 mg / mL) solution in PBS, delivered at a volume of 0.1 mL per 10 grams of body weight. Subretinal injections were carried out under a surgical microscope (Zeiss). A comeal incision was created in the posterior segment of the right eye using a 31 -gauge needle. A pre-pulled glass micropipette (Fivephoton Biochemicals, MGM-1D) was then inserted through this incision. The micropipette, which was coupled to a 1 mL syringe (BD, 309623) via the tubing from a butterfly infusion set. was advanced through the retinal layers. A total volume of 1.5 pL of AAV, at a concentration of 5 x 1012vg / mL, was injected into the subretinal space.

[0198] Isolating transduced retinal cells Retinal tissue was harvested from mice and subjected to microdissection. A homogeneous single-cell suspension was generated through enzymatic dissociation using a papain-based kit, following the supplier's protocol (Worthington Biochemical. Lome Laboratories). The resulting cell suspension was washed and resuspended in HBSS. To distinguish viable cells, SYTOX Blue Dead Cell Stain (Thermofisher, S34857) was added. Samples were maintained on ice and subsequently subjected to flow cytometric analysis and sorting on a FACSAria instalment (BD Biosciences) to isolate live, GFP-positive cells.

[0199] Quantifying in vivo genome editing Genomic DNA (gDNA) was isolated from the FACS-sorted, GFP-positive retinal cells to quantify editing efficiency. Sorted cells were pelleted and lysed in a buffer containing 10 mM Tris, 10 mM EDTA, 10 mM NaCl, 0.5% Sarcosyl, andCOLUM-44474.6010.4 mg / mL proteinase K. The lysis reaction was incubated at 56°C for 2 hours, followed by heat inactivation at 95°C for 30 minutes.

[0200] The targeted genomic region surrounding the SAG p.C147F locus was then amplified from the resulting gDNA lysate. PCR was performed using primers containing partial Illumina adapter sequences and SuperFi II polymerase, following the protocol established for in vitro analysis. The amplicons were purified and prepared for next- generation sequencing as previously described. Sequencing was performed by Genewiz (Amplicon-EZ service), and the resulting data were analyzed using Cas-Analyzer to determine the frequency of indels.Example 1

[0201] Cas nucleases were transfected into p.C147F SAG HEK293T cells which comprise the nucleic acid sequence of TTTG, having the PAM for Casl2j nucleases allowing monitoring of indels in the gene as a result of the activity of Casl2j. As shown in FIG. 1, wild-type Casl2j results in indel formation in p.C147F SAG HEK293T cells. FIGS. 2A and 2B show the indel formation single amino acid variants and multiple amino acid variants in wild-type Casl2j. v3.5 Casl2j (corresponding to SEQ ID NO: 2) is shown boxed in FIG. 2B.

[0202] Direct comparison between wild-type Casl2j and v3.5 Casl2j (also referred to as CasPhiX and Casl2j v3.5) is shown in FIG. 3. Notably, v3.5 Casl2j results in significantly more percent indels than wild-type Casl2j. The comparison was made across an additional twelve sites (FIGS. 4A and 4B) and a further six sites (FIGS. 4C and 4D). Overall, Casl2j resulted in more percent indels than wild-type Casl2j.Example 2

[0203] The SAG p.C147F (G>T) mutation causes autosomal dominant retinitis pigmentosa (adRP) through a proposed gain-of-function mechanism. Previous research indicates the SAG gene is haplosufficient, making an allele-specific knockout of the disease allele a viable therapeutic strategy. SAG p.C147F is a single point mutation (G>T) that creates a novel PAM site (5’-TTTR-3’) which is targetable by many class II type V Casl2 orthologs. Multiple compact Cas 12 nucleases were screened for an allele-specific therapeutic strategy (FIG. 6A). A homozygous SAG p.C147F HEK293T cell line was created using prime editing. Four type V Casl2 orthologs: AsCasl2f (422 aa), AsCasl2a (1307 aa), CasMINI (529 aa), and Casl2j (717 aa) were transfected into homozygous SAG p.C147F HEK293T cells. Amplicon-based deep sequencing revealed that only Casl2j and AsCasl2a led to noticeable indels (20.47 ± 2.46 %COLUM-44474.601Casl 2j, 54.66 ± 6.25% AsCasl2a, respectively, p<0.0001, FIG. 6B). However, AsCasl2a’s large size (1307 amino acids) precludes single adeno-associated viral (AAV) vector packaging (FIG.6C). Casl2j’s compact size (717 amino acids) does allow for single AAV vector packaging, but the nuclease showed limited editing efficiency.Example 3

[0204] As described in Example 2, the compact size of Casl2j is advantageous for AAV packaging but its editing efficiency targeting SAG p.C147F was suboptimal for therapeutic application. A structure-guided protein engineering approach was used to enhance its nuclease activity. Multiple sequence alignments of Casl2j with other Casl 2 nucleases were used to identify conserved functional domains (FIG. 7A). This was integrated with a structural model predicted by AlphaFold3 of Casl2j targeting SAG p.C147F. Conserved amino acids within DNA or crRNA binding domains were targeted for engineering.

[0205] Focusing on substituting negatively charged or neutral residues with arginine or lysine to improve binding affinity, a library of 60 Casl2j variants was constructed and screened for activity in homozygous SAG p.C147F HEK293T cells (FIG. 7B). From the initial screen, five candidates showed improvement over Casl2j (L26K, A121Q, N147K, S186G, and E258K) with all but N147K showing greater than 1.5-fold improvement over WT Casl2j (FIG. 7B). Ten paired combinations of these five variants were generated to screen for additive beneficial mutations (v2), and six triple mutation variants (v3) (FIG. 7C). Casl2j v3.5 (L26K, N147K, and E258K) was identified as the top candidate showing greater than 3.3-fold improvement over WT Casl2j. To confirm allele-specificity was maintained, Casl2j v3.5 was screened in wild-type SAG HEK293T cells. Casl2j v3.5 was able to target WT SAG in HEK293T, with an average of 20.1% indels (FIG. 7D). Through improving DNA and crRNA binding affinity, the PAM specificity of Casl2j was inadvertently reduced.Example 4

[0206] As described in Example 3, despite its enhanced potency, the broadened PAM recognition of Casl2j v3.5 resulted in unintended editing of the wild-type SAG allele. To overcome this lack of specificity, mismatches in the crRNA spacer were introduced to screen the impact on editing efficiency and specificity, such that the SAG p.C147F disease allele is ablated while the WT SAG allele is preserved.COLUM-44474.601

[0207] Due to the PAM preference of Casl2j (5’-TTN-3’), there are two different crRNA spacer options, differing only by a single base pair shift (FIG. 8A). Previous engineering efforts and optimizations were conducted using crRNA 1, single nucleotide mismatches were systematically introduced at each position within crRNA 1. These mismatched crRNA spacers were tested in both SAG p.C147F and WT HEK293T cells (FIGS. 5A and 5B). This comprehensive profiling allowed identification of specific positions where a mismatch was tolerated by Casl2j v3.5 in the SAG p.C147F cells but not in the WT cells. Of particular note, mismatches in position 9, 16, and 17 showed >5% indels in SAG p.C147F cells but with severely reduced indels in WT cells (<0.25%). A mismatch in position 18 did not reduce editing compared to no mismatches, indicating this position seems to be tolerated by both WT and Casl2j v3.5 (e.g. Casl2j v3.5: 71.37 ± 10.68% no mismatch, 71.57% ± 8.92% with a mismatch). However, a mismatch in position 17 resulted in 23.3% indels targeting SAG p.C147F, and 0.23% targeting WT SAG using Casl2j v3.5 demonstrating high specificity but less than desired ablation of the disease (FIGS. 5A and 5B).

[0208] Given the lack of a modified crRNA 1 that shows high editing efficiency targeting SAG p.C 147F while maintaining high specificity, all possible mismatches were installed at position 16 and 17 of crRNA spacer 2. The introduction of mismatches in crRNA spacer 2 showed great promise, with mismatch 17 (T>G) demonstrating strong ablation in SAG p.C147F cells, but almost no editing in WT HEK293T cells (53.1 ± 13.6% and 0.33 ± 0.19%, respectively, FIGS. 8B and 8C). Even without the introduction of mismatches, Casl2j v3.5 with crRNA spacer 2 showed strong allele-specificity (62.05 + 13.76% SAG p.C147F, 3.08 ± 1.96% SAG WT, FIGS. 8B and 8C). crRNA spacer 2 with mismatch 17 (T>G) was used to future studies.Example 5

[0209] To evaluate the therapeutic potential of the optimized system, two humanized mouse models of SAG were developed by replacing the endogenous mouse SAG exon 7 with the humanized WT sequence and one with the p.C147F human sequence (FIG. 9A). Casl2j v3.5 and the allele- specific crRNA (spacer 2 with mismatch 17 (T>G)) were packaged into a single AAV vector (FIG. 9B). This all-in-one vector was delivered via subretinal injection to humanized homozygous SAG mice bearing either the pathogenic SAG p.C147F mutation or the wild-type SAG allele.COLUM-44474.601

[0210] Quantitative autofluorescence (qAF) imaging confirmed robust and widespread transduction of the retina in injected mice (FIG. 9C). Analysis of genomic DNA extracted from transduced retinal cells revealed efficient editing in homozygous SAG p.C147F mice, with indels reaching up to 54.6% and a mean of 45.46 ± 10.5% (FIG. 9D). Encouragingly, editing in humanized SAG WT mice was minimal (<0.01%, FIG. 9D). The high allele-specificity observed in vitro (> 100-fold preference for p.C147F allele) was maintained in the in vivo environment (Fig. 4d).Example 6

[0211] Having demonstrated Casl2j v3.5 increased indels when targeting SAG p.C147F, the editing efficiency of Casl2j v3.5 was compared to other compact Gas 12 nucleases (CasMINI ge_v4.1 (529 aa), AsCasl2f v5.1 (422 aa), hfCasl2Max (1081 aa), and the canonical larger AsCasl2a (1307 aa)), using spacers without mismatches. Twelve sites were chosen that could accommodate the PAM preference of each nuclease (FIG. 10A).

[0212] Across the twelve sites Casl2j v3.5 outperformed Casl2j at eleven sites (all but PDCD1), showing very high editing at some sites (82.58 ± 4.45% targeting PCSK9 v.s. 33.4 ±0.31% for WT Casl2j). Casl2j v3.5 induced more indels at nine sites compared to AsCasl2f v5.1, and at 11 sites compared to CasMINI ge_v4.1. However, hfCasl2Max outperformed Casl2j v3.5 at nine out of twelve sites. AsCasl2a outperformed Casl2j v3.5 at one site, while Casl2j v3.5 outperformed AsCasl2a at two sites, with the rest showing no significant difference in efficiency (FIG. 10A). Across the twelve sites, Casl2j showed mean editing efficiencies of 25.71 ± 8.87%, Casl2j v3.556.89 ± 13.42%, AsCasl2f v5.1 25.38 ± 16.44%, CasMINI ge_v4.1 11.59 ± 9.67%, hfCasl2Max 82.06 ± 7.48%, and AsCasl2a 56.28 ± 13.35% indels (FIG. 10C). Overall Casl2j v3.5 showed improved efficiency compared to Casl2j, AsCasl2f v5.1, CasMINI ge_v4.1, was comparable to the larger AsCasl2a. but hfCasl2Max showed greater indel formation than Casl2j v3.5.

[0213] Casl2j v3.5 was compared to SpCas9 across seven sites. To overcome SpCas9 and Casl2j v3.5 differences in PAM preference and orientation, sites where spacers overlapped were chosen to conduct the comparison. Across the sites, SpCas9 outperformed Casl2j v3.5 at two sites, was outperformed by Casl2j v3.5 at one site, and the rest showed no difference in editing efficiency (FIG. 10B). Casl2j showed mean indels of 22.53 + 12.54%, Casl2j v3.5 49.79 ±COLUM-44474.60121.57%, and SpCas954.12 ± 24.23% across all sites (FIG. 10D). For the few genomic loci selected Casl2j v3.5 showed comparable efficiency to SpCas9, with two exceptions, demonstrating its strong activity and potential (two-tail paired t-test p = 0.7431).Example 7

[0214] Mouse models of C110R rhodopsin were made by installing the T>C mutation by Prime Editing. Validations of ablation of Cl 10R was completed using wild-type Casl2j, v3.5 Casl2j (also referred to as CasPhiX and Casl2j v3.5), and hfCasl2Max with crRNA spacers of gggcccacaggaCgcaat and cGtcctgtgggcccgaag (SEQ ID NOs: 71 and 72, respectively), represented as DNA, is shown in FIG. 11. Notably, for this target, as compared to SAG allele, the crRNA spacer sequences without mismatches were allele-specific. Contrarily, hfCasl2Max is not allele- specific for RHO p.Cl 10R.

[0215] An AAV encoding v3.5 Casl2j with crRNA spacer was designed, as shown in FIG.12. Heterozygous Cl 10R RHO mice are injected with the AAV to characterize functional rescue by electroretinogram (ERG) and histopathology.SequencesWT Casl2j or CasPhi (SEQ ID NO: 1) MIKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIA NIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEAA GLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFEEIKAFDDKGY LLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPIGEP GYVPKWQYTFLSKKENKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHWKKY HKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICDQN GSCKLATVDVGQNNPVAIGLFELKKVNGELTKTLISRHPTPIDFCNKITAYRERYDKLES SIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLNINPNDLPWDKMISGTHFISEKA QVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEF NKLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFGGSGKREPGWDNFYKPKKENR WWINAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELN ADIDVATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDKLAPSYTVVLRE AVEngineered v3.5 Casl2j (also referred to as Casl2j-8 v3.5 or CasPhiX) (SEQ ID NO: 2) MIKPTVSQFLTPGFKLIRNHSRTAGKKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIA NI1AKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEAA GLNLIIKNAVNTYKGVQVKVDNKNKKNLAKINRKNEIAKLNGEQEISFEEIKAFDDKGY LLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPIGEPCOLUM-44474.601GYVPKWQYTFLSKKKNKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHWKKY HKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICDQN GSCKLATVDVGQNNPVAIGLFELKKVNGELTKTLISRHPTPIDFCNKITAYRERYDKLES SIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLNINPNDLPWDKMISGTHFISEKA QVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLSKEVRDALSDIEWRLRRESLEF NKLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFGGSGKREPGWDNFYKPKKENR WWINAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKNRNGEKFNCLKCGIELN ADIDVATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDKLAPSYTVVLRE AVcrRNA 1 targeting SAG p.C147F (represented as DNA)crRNA 2 targeting SAG p.C147F (represented as DNA)COLUM-44474.601crRNA (sequence represented as DNA)

[0216] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions.COLUM-44474.601Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0217] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

COLUM-44474.601CL IMSWhat is claimed is:

1. An engineered nuclease comprising less than 100% amino acid sequence identity with wildtype Casl2j (SEQ ID NO: 1) and at least one amino acid substitution compared to SEQ ID NO: 1,wherein the engineered nuclease has increased efficiency as compared to wild-type Casl2j, andwherein the at least one amino acid substitution comprises an amino acid substitution at positions 26, 147, 258, or a combination thereof, relative to SEQ ID NO: 1.

2. The engineered nuclease of claim 1, wherein the at least one amino acid substitution comprises amino acid substitutions at positions 26, 147, and 258.

3. The engineered nuclease of claim 1 or 2, wherein the at least one amino acid substitution comprises substitution with a positively charged amino acid.

4. The engineered nuclease of any of claims 1-3, wherein the engineered nuclease comprises an L26K, N147K, and / or E258K amino acid substitution with reference to SEQ ID NO: 1.

5. The engineered nuclease of any of claims 1-4, wherein the engineered nuclease has an amino acid sequence with at least 75%, at least 80%. at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 2.

6. The engineered nuclease of any of claims 1-5, wherein the engineered nuclease has an amino acid sequence of SEQ ID NO: 2.

7. The engineered nuclease of any of claims 1-6, wherein the engineered nuclease further comprises a localization sequence, a tag sequence, a protein transduction domain sequence, or a combination thereof.

8. A fusion protein comprising an engineered nuclease of any of claims 1-7 and an effector domain.

9. A system or kit for nucleic acid modification comprising:COLUM-44474.601an engineered nuclease as in any of claims 1-7, a fusion protein comprising the engineered nuclease, or a nucleic acid encoding the engineered nuclease or fusion protein; and at least one gRNA complementary to at least a portion of a target nucleic acid sequence, or a nucleic acid encoding the at least one gRNA.

10. The system or kit of claim 9, wherein:the at least one gRNA is encoded by a nucleic acid different from the nucleic acid encoding the engineered nuclease or fusion protein;the at least one gRNA is encoded by a nucleic acid also encoding the nucleic acid encoding the engineered nuclease or fusion protein; and / orthe at least one gRNA is complexed with the engineered nuclease or fusion protein.

11. A composition comprising an engineered nuclease as in any of claims 1-7, a fusion protein comprising the engineered nuclease, a nucleic acid encoding the engineered nuclease or fusion protein, or a system of claim 9 or 10.

12. A cell comprising an engineered nuclease as in any of claims 1-7, a fusion protein comprising the engineered nuclease, a nucleic acid encoding the engineered nuclease or fusion protein, or a system of claim 9 or 10.

13. A method for nucleic acid modification comprising contacting a target nucleic acid sequence with an engineered nuclease as in any of claims 1-7, a fusion protein comprising the engineered nuclease, a nucleic acid encoding the engineered nuclease or fusion protein, a system of claim 9 or 10, or a composition thereof.

14. The method of claim 13, wherein the target nucleic acid sequence is in a cell and contacting a target nucleic acid sequence comprises introducing the system into the cell.

15. The method of claim 14, wherein introducing the system into the cell comprises administering the system to a subject.

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