Engineered nucleases and compositions and methods thereof

Abstract

The present disclosure provides engineered nucleases and compositions, methods, and systems thereof for nucleic acid modification. More particularly, the present disclosure provides engineered nucleases comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 2-16 and / or having at least 70% identity to SEQ ID NO:1 with one or more additions, substitutions, or deletions at positions 3, 18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465, relative to SEQ ID NO:1.

Description

ENGINEERED NUCLEASES AND COMPOSITIONS AND METHODS THEREOFFIELD

[0001] The present invention relates to engineered nucleases and compositions, methods, and systems thereof for nucleic acid modification.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 587,321, filed October 2, 2023, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled ACRIG_42448_601_SequenceListing.xml (Size: 49,942 bytes; and Date of Creation: September 24, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases dominate the nucleic acid-editing landscape because they are versatile, rapid, and easy-to-use editing tools. The most well-characterized CRISPR-Cas nuclease, Cas9, utilizes one or more RNAs to act as a sequence-specific targeting element linking the nuclease to the target nucleic acid. However, presently CRISPR / Cas systems have some limitations for use, particularly in eukaryotic organisms including low efficiency of editing, off-target events, target sequence preferences and efficient delivery and expression of the nuclease.SUMMARY

[0005] Provided herein are engineered nucleases having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity with any one of SEQ ID NOs: 2-16. In some embodiments, the engineered nuclease has one or more amino acid substitution, deletion, or addition as compared to the amino acid sequence of SEQ ID NO: 1.

[0006] Provided herein are engineered nuclease s having at least 70% sequence identity with SEQ ID NO: 1 and comprising at least one amino acid substitution, deletion or addition at a position selected from: 3, 18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465 relative to SEQ ID NO:1. In some embodiments, the engineered nuclease includes 2, 3, 4, 5, 6, 7 or more than 7 amino acid substitutions at positions selected from the group consisting of: 3,18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465 relative to SEQ ID NO: 1.

[0007] In some embodiments, the engineered nuclease comprises an amino acid modification at position 141 relative to SEQ ID NO:1. In select embodiments, the substitution at position 141 is H141Q.|0008] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at position 184. In select embodiments, the substitution at position 184 is Q184H.

[0009] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at (a) position 141, (b) position 379 or 380 and (c) position 462 or 463. In select embodiments, the substitution at position 141 is H141Q, the substitution at positions 379 and 380 is D379G or E380V and the substitution at position 462 or 463 is E462D or G463A.

[0010] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 90, 141, and 184. In select embodiments, the substitutions are D90V, H141Q, and Q184H.

[0011] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 141, 165 and 379. In select embodiments, the substitutions are H141Q, N165Y, and D379G.

[0012] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 141, 148 and 251. In select embodiments, the substitutions are H141Q, Q148H, and V251L.

[0013] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 141, 148, 251, 380, and 463. In select embodiments, the substitutions are H141Q, Q148H, V251L, E380V, and G463A.

[0014] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 40, 141, 148, 251, 283, and 312. In select embodiments, the substitutions are N40Y, H141Q, Q148H, V25 IL, N283Y, and R312Q.|0015] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 90, 141, 379, and 462. In select embodiments, the substitutions are D90V, H141Q, D379G, and E462D.

[0016] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 141, 184, 379, and 463. In select embodiments, the substitutions are H141Q, Q184H, D379G, and G463A.|0017] In some embodiments, the engineered nuclease comprises an amino acid substitution, deletion and / or addition at positions 40, 141, 155, 380, and 463. In select embodiments, the substitutions are N40Y, H141Q, S155G, E380V, and G463A.

[0018] In some embodiments, the engineered nuclease comprises the amino acid sequence of any of SEQ ID NOs: 2-16.

[0019] In some embodiments, the engineered nuclease is modified in its PAM preference as compared to the PAM preference of SEQ ID NO: 1.

[0020] In some embodiments, the engineered nuclease further contains one or more substitutions, additions, or deletions that inactivate the cleavage function of the nuclease. In some embodiments, the engineered nuclease retains the ability to bind to a target DNA.

[0021] In some embodiments, the engineered nuclease further comprises a nuclear localization sequence (NLS).

[0022] Also provided herein are fusion proteins comprising an engineered nuclease as described herein covalently linked to a second protein or protein domain.

[0023] In some embodiments, the second protein or protein domain comprises an adenosine deaminase or a cytidine deaminase. In some embodiments, the second protein or protein domain comprises one or more catalytic domains of the adenosine deaminase (TadA domain). In some embodiments, the TadA domain comprises any of SEQ ID NOs: 20-23. In some embodiments, the fusion protein comprises any of SEQ ID NOs: 17-19.

[0024] Further provided are nucleic acids encoding an engineered nuclease or fusion protein as described herein. In some embodiments, the nucleic acid is codon-optimized for expression of the engineered nuclease or the fusion protein in a eukaryotic cell. In some embodiments, the nucleic acid is codon-optimized for expression of the engineered nuclease or the fusion protein in a mammalian cell.

[0025] Additionally provided is an expression cassette comprising a promoter operatively linked to a nucleic acid as described herein and vectors comprising the nucleic acid or expression cassette.|0026] Provided herein are systems. In some embodiments, the systems are configured to modify a target nucleic acid. In some embodiments, the systems comprise an engineered nuclease, a fusion protein, a nucleic acid, an expression cassette, or a vector as described herein and at least one guide RNA (gRNA). In some embodiments, the gRNA is an engineered gRNA comprising a first region configured to interact with the engineered nuclease and a second region configured to interact with the target.|0027] In some embodiments, the target nucleic acid comprises a nucleic acid sequence region within a mammalian genome. In some embodiments, the nucleic acid sequence region is a gene, a regulatory region, an intron, a splice site, or an intergenic region. In some embodiments, the engineered nuclease in the presence of the gRNA is capable of modifying the target nucleic acid.|0028] In some embodiments, modifying the target nucleic acid comprises one or more of nucleic acid binding, nucleic acid cleavage, base editing, transcriptional modification, and histone modulation.

[0029] In some embodiments, the system comprises a first nucleic acid comprising the nucleic acid encoding the engineered nuclease or the fusion protein and a second nucleic acid comprising the gRNA or a nucleic acid encoding the gRNA. In some embodiments, the first nucleic acid and the second nucleic acid are comprised on a single expression cassette or vector. In some embodiments, the first nucleic acid and the second nucleic acid are comprised on separate expression cassettes or vector. In some embodiments, the first nucleic acid is operatively linked to a first promoter and the second nucleic acid is operatively linked to a second promoter. In some embodiments, the first promoter and / or the second promoter are active in a mammalian cell. In some embodiments, the first promoter and / or the second promoter are active in a human cell. In some embodiments, the first promoter and the second promoter are the same. In some embodiments, the first promoter and the second promoter are different. In some embodiments, the first nucleic acid and / or the second nucleic acid are comprised on a viral vector.

[0030] In some embodiments, the system comprises one or more cells. In some embodiments, the system is cell-free.

[0031] Also provided are cells comprising an engineered nuclease, a fusion protein, a nucleic acid, an expression cassette, a vector, or a system as 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. In some embodiments, the cell is a human cell.

[0032] Further provided is an in vitro assay comprising a system as disclosed herein.|0033] Provided are methods of modifying a target nucleic acid sequence. In some embodiments, the methods comprise contacting the target nucleic acid with a targeting composition comprising an engineered nuclease, fusion protein, nucleic acid, expression cassette, vector, or system as described herein.|0034] In some embodiments, the target nucleic acid sequence is in a cell. In some embodiments, the cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is a human cell. In someembodiments, contacting a target nucleic acid sequence comprises introducing the targeting composition into the cell. In some embodiments, contacting a target nucleic acid sequence comprises administering the targeting composition to a subject.

[0035] In some embodiments, the target nucleic acid sequence encodes a gene product.|0036] In some embodiments, the targeting composition comprises at least one non-naturally occurring gRNA.

[0037] Also provided are methods for treating a disease or disorder in a subject. In some embodiments, the methods comprise administering to the subject in need thereof a targeting composition comprising an engineered nuclease, fusion protein, nucleic acid, expression cassette, vector, or system as described herein and at least one guide RNA (gRNA) complementary to at least a portion of a target nucleic acid or a nucleic acid comprising a sequence encoding the at least one gRNA. In some embodiments, the target nucleic acid is a disease-associated gene. In some embodiments, the subject is a human.

[0038] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is a graph of the editing activity of EMX1 and FANCF in HEK293T for the nuclease variants of SEQ ID NO: 1.

[0040] FIG. 2 is a table of the base editing levels of adenine base editor constructs (SEQ ID NOs: 21- 23), shown as a percentage of editing at each position in the sequence covered by the guide.DETAILED DESCRIPTION

[0041] The disclosed compositions, systems, kits, and methods comprise engineered nucleases useful for nucleic acid modification. The disclosed nucleases allow for gene editing with improved efficacy and safety for use in in vivo and ex vivo applications, including such application for eukaryotic (e.g., mammalian (e.g., human)) therapeutics, diagnostics, and research.

[0042] 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

[0043] 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.

[0044] 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.

[0045] 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, microbiology, 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.

[0046] 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 nonnucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used 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 double-stranded, 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.

[0047] 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(11): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0048] 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 atleast 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.|0049] 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.

[0050] 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.

[0051] 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.

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

[0053] 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 byreference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Engineered Nucleases

[0054] Advances and developments in CRISPR-Cas genome editing tools including nucleases and other Cas protein drive major advances in nucleic acid editing. Nucleic acid editing has many uses including in the diagnostics and therapeutics field. Such breadth is accompanied by a diversity of nucleic acid targets and environments in which to engineer editing activity. As such, there is a need for diverse and additional nucleases and associated methods that provide a toolbox for nucleic acid editing.|0055] Disclosed herein are engineered nucleases comprising a sequence having at least 70% identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to any one of SEQ ID NOs: 2-16. In some embodiments, the engineered nucleases comprise an amino acid sequence of any of SEQ ID NOs: 2-16.

[0056] Disclosed herein are engineered nucleases comprising a sequence having at least 70% identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to an amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered nucleases do not comprise SEQ ID NO: 1.

[0057] In some embodiments, the engineered nucleases include one or more amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the engineered nucleases comprise at least one amino acid substitution, deletion or addition at a position selected from: 3, 18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465 relative to SEQ ID NO: 1. In some embodiment, the engineered nuclease comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 2, 3, 4, 5, 6, 7 or more than 7 amino acid substitutions at positions selected from the group consisting of: 3, 18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465 relative to SEQ ID NO: 1. For example, the engineered nuclease may comprise substitutions at positions: 90 and 141; 40 and 141;40, 141, and 155; 141 and 184; 141 and 380; 90, 141, and 184; 141, 148, 251, 380, and 463; 404, 141, 148, 251, 283, and 312; 90, 141, 379, and 462; 40, 141, 155, and 398; 40, 141, 155, 304, 363, 462; 141, 155, and 398; 141, 184, 379, and 463; 18, 141, and 465; 141, 155, 304, 363, and 462; 3, 40, 141, 253, and 398; 18, 141, 184, 367, and 465; 3, 41, 90, 122, 141, and 379; 40, 141, 155, 380, and 463; and 141, 165, and 379.

[0058] In some embodiments, the engineered nucleases comprise at least one amino acid substitution, deletion, or addition at position 141 relative to SEQ ID NO: 1. In some embodiments, the engineered nucleases comprise an amino acid substitution at position 141 relative to SEQ ID NO: 1 and optionally, at least one additional substitution, relative to SEQ ID NO:1. In select embodiments, the substitution at position 141 is H141Q.

[0059] In some embodiments, the engineered nucleases comprise at least one amino acid substitution, deletion, or addition at position 184 relative to SEQ ID NO: 1. In some embodiments, the engineered nucleases comprise an amino acid substitution at position 184 relative to SEQ ID NO: 1 and optionally, at least one additional substitution, relative to SEQ ID NO:1. In select embodiments, the substitution at position 184 is H184Q.

[0060] In some embodiments, the engineered nucleases comprise at least one amino acid substitution, deletion, or addition at position 379 or 380. In select embodiments, the substitutions at position 379 is D379G. In select embodiments, the substitutions at position 380 is E380V.

[0061] In some embodiments, the engineered nucleases comprise at least one amino acid substitution, deletion, or addition at position 462 or 463. In select embodiments, the substitutions at position 462 is E462D. In select embodiments, the substitution at position 463 is G463A.

[0062] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 90, 141, and 184. In select embodiments, the engineered nuclease has the substitutions D90V, H141Q, and Q184H.

[0063] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 141, 165 and 379. In select embodiments, the engineered nuclease has the substitutions H141Q, N165Y, and D379G.

[0064] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 141, 148 and 251. In select embodiments, the engineered nuclease has the substitutions H141Q, Q148H, and V251L.|0065] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 141, 148, 251, 380, and 463. In select embodiments, the engineered nuclease has the substitutions H141Q, Q148H, V251L, E380V, and G463A.

[0066] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 40, 141, 148, 251, 283, and 312. In select embodiments, the engineered nuclease has the substitutions N40Y, H141Q, Q148H, V251L, N283Y, and R312Q.

[0067] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 90, 141, 379, and 462. In select embodiments, the engineered nuclease has the substitutions D90V, H141Q, D379G, and E462D.

[0068] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 141, 184, 379, and 463. In select embodiments, the engineered nuclease has the substitutions H141Q, Q184H, D379G, and G463A.

[0069] In some embodiments, the engineered nuclease has substitutions, deletions, and / or additions at positions: 40, 141, 155, 380, and 463. In select embodiments, the engineered nuclease has the substitutions N40Y, H141Q, S155G, E380V, and G463A.

[0070] Any of the engineered nucleases described herein may comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 150, etc.) amino acid substitutions as compared to SEQ ID NO: 1 in addition to those expressly recited above. 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), tyrosine (Y or Tyr), and tryptophan (W or Trp). Nonaromatic 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 (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0071] The amino acid replacement or substitution can be conservative, semi-conservative, or nonconservative. 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 ofamino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Spring er- 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 subgroup. 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.

[0072] In some embodiments, the engineered nuclease comprises one or more substitutions, deletions, and / or additions which result in at least partial inactivation of the nuclease catalytic activity. For example, in instances when the nuclease comprises two catalytic sites, one or both of the catalytic site may include a substitution, deletion, and / or addition which results in suppressed or abolished activity. In select embodiments, the engineered nuclease is fully catalytically inactivated. In some embodiments, the engineered nuclease comprises substitutions at positions 269 and 467, relative to SEQ ID NO: 1. In select embodiments, the engineered nuclease comprises substitutions D269A and D467A, relative to SEQ ID NO: 1.

[0073] In some embodiments, abolishing the nuclease activity retains the DNA binding activity of the engineered nuclease. Accordingly, fully or partially catalytically inactivated engineered nucleases as disclosed herein may still function as a DNA binding protein, with the targeting capabilities of the fully functioning nuclease.

[0074] The engineered nucleases disclosed herein may be modified in its protospacer adj acent motifs (PAM) preference as compared to the PAM preference of SEQ ID NO: 1. In certain embodiments, the engineered nuclease can only cleave a target nucleic acid if an appropriate PAM is present. PAM sequences and methods of determining PAM sequences for specific proteins are known in the art.|0075] A PAM is generally in proximity to a target sequence. For example, the PAM may be a sequence immediately or directly adjacent to the target nucleic acid. 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 nucleic acid is immediately flanked on the 3’ end by a PAM. In one embodiment, the target nucleic acid is immediately flanked on the 5’ end by a PAM. 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.

[0076] In some embodiments, the engineered nuclease further comprises a nuclear localization sequence (NLS). The nuclear localization sequence may be appended, for example, to one or both of the N-terminus and C-terminus. In some embodiments, the engineered nuclease comprises two or more NLSs. The two or more NLSs may be in tandem, separated by a linker, at either the N-terminus or C- terminus of the protein, or one or more may be internal to the open reading frame of the engineered nuclease.

[0077] 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.

[0078] 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 NLS sequences include those from the SV40 large T-antigen, c-Myc, and TUS-proteins. In select embodiments, the NLS comprises the NLS of SV40 large T-antigen, comprising an amino acid sequence of PKKKRKV (SEQ ID NO: 42).

[0079] 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 nuclear localization sequences of nucleoplasmin, EGL-12, or bipartite SV40. In select embodiments, the NLS comprises the NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 43).

[0080] In some embodiments, the two or more NLSs may have the same or different sequences. For example, in some embodiments, the engineered nuclease comprises two NLSs, one sequence from the SV40 large T-antigen and one from nucleoplasmin.|0081] The NLS may be appended to the engineered nuclease by a linker. The linker may have any of a variety of amino acid sequences. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. Small amino acids, such as glycine and alanine, are generally used in creating a flexible peptide. A variety of different linkers are commercially available and are considered suitable for use, including but not limited to, glycine-serine polymers, glycine-alanine polymers, and alanine-serine polymers.

[0082] In some embodiments, the engineered nuclease may comprise a tag (e.g., 3xFLAG tag, an HA tag, a Myc tag, and the like). The tag may facilitate tracking, separation, or purification of the engineered nuclease. In some embodiments, the tag may be adjacent, either upstream or downstream, to a nuclear localization sequence. The tag may be at the N-terminus, a C-terminus, or a combination thereof of the engineered nuclease.

[0083] In some embodiments, the engineered nuclease is covalently attached to a peptide or protein in a fusion protein. The engineered nuclease may be covalently attached to a peptide or protein with a linker as described above.

[0084] In some embodiments, the engineered nuclease may be fused with one or more (e.g., two, three, four, or more) protein transduction domains or PTDs, also known as a CPP - cell penetrating peptide. A protein transduction domain is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to a terminus of the engineered nuclease (e.g., N-terminus, C-terminus, or both). In some embodiments, the PTD is inserted internally at a suitable insertion site. Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52 T) V132' -1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); Transportan, and the like.

[0085] The engineered nuclease may be part of a fusion protein comprising another protein or protein domain. For example, the engineered nuclease may be fused to another protein or protein domain that provides for tagging or visualization (e.g., GFP). The engineered nuclease may be fused to a protein or protein domain that has another functionality or activity useful to target to certain DNA sequences (e.g., engineered nuclease activity such as that provide by FokI engineered nuclease, protein modification activity such as histone modification activity including acetylation or deacetylation or demethylation or methyltransferase activity, transcription modulation activity such as activity of a transcriptional activator or repressor, base editing activity such as deaminase activity, DNA modifying activity such as DNA methylation activity, and the like).

[0086] Also provided herein are fusion proteins comprising an engineered nuclease, as disclosed herein, fused to a deaminase, or functional fragment thereof. A deaminase, or a functional fragment thereof, is a protein, enzyme, domain, or moiety which catalyzes a deamination reaction. The deaminase, or functional fragment thereof may be derived from a naturally-occurring deaminase or variant thereof (e.g., a protein, enzyme, or domain with an amino acid sequence having at least 70% identity to a naturally-occurring deaminase). Alternatively, the deaminase may be a synthetic or engineered deaminase.

[0087] In some embodiments, the deaminase, or functional fragment thereof, is an adenosine deaminase, also sometimes referred to as an adenine deaminase. Adenosine deaminases catalyze the deamination of adenosine and deoxyadenosine to inosine and deoxyinosinse, respectively. Accordingly, with repair and replication mechanisms adenosine deaminase can ultimately lead to the conversion of an A:T nucleobase pair to a G:C nucleobase pair.

[0088] In some embodiments, the adenosine deaminase is derived from a bacterium, such as, E. coli. In some embodiments, the adenosine deaminase is a TadA deaminase or functional variant or fragment thereof. In some embodiments, the TadA deaminase is an E. coli TadA deaminase. In some embodiments, the TadA deaminase is a truncated TadA deaminase. In some embodiments, the adenosine deaminase comprises one or more catalytic domains or TadA domains.|0089] In some embodiments, the TadA domain comprises any of SEQ ID NOs: 20-23. In some embodiments, the fusion protein comprises one or more of SEQ ID NOs: 20-23 and an engineerednuclease sequence, as described herein. For example, the fusion protein may comprise an engineered nuclease having at least 70% identity to any of SEQ ID NOs: 1-16 and one or more of SEQ ID NOs 20- 23. In some embodiments, the engineered nuclease comprises at least one amino acid substitution, deletion or addition at a position selected from: 3, 18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465 relative to SEQ ID NO: 1. In some embodiments, the engineered nuclease further contains one or more mutations or deletions that inactivate the cleavage function of the nuclease. In some embodiments, the fusion protein comprises any of SEQ ID NOs: 17-19.

[0090] In some embodiments, the deaminase, or functional fragment thereof, is a cytidine deaminase. Cytidine deaminases catalyze the deamination cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Cytidine deaminases may also catalyze the reaction of 5 -methyl cytosine to thymine. Accordingly, with repair and replication mechanisms a cytidine deaminase can ultimately lead to the conversion of a C to a U or T. With repair and replication mechanisms adenosine deaminase can ultimately lead to the conversion of a C:G nucleobase pair to a U / T:A nucleobase pair. Cytidine deaminases include, but are not limited to members of the APOBEC family, e.g., APOBEC1, and activation-induced cytidine deaminase (AID).Compositions and Systems

[0091] Also disclosed herein are systems and compositions comprising an engineered nuclease or fusion protein as described herein or a nucleic acid encoding the engineered nuclease or fusion protein.

[0092] In some embodiments, the systems are applicable for modifying a target (e.g., a target nucleic acid). The modifying may include one or more of: nucleic acid binding, nucleic acid cleavage, base editing, transcriptional modification, and histone modulation. In some embodiments, the target is a nucleic acid sequence region within a mammalian genome. In some embodiments, the nucleic acid sequence region is a gene, a regulatory region, an intron, a splice site, or an intergenic region.

[0093] The compositions and systems may further comprise at least one gRNA comprising a sequence complementary to at least a portion of a first target nucleic acid and a region that associates with the engineered nuclease, or a nucleic acid encoding the at least one gRNA. In some embodiments, the at least one gRNA further comprises a sequence complementary to at least a portion of a second target nucleic acid. In instances when the composition or system comprises more than one gRNA, each may be encoded on the same or different nucleic acid as the other gRNA.|0094] The gRNA may be a crRNA, crRNA / tracrRNA (or single guide RNA, sgRNA). The terms “gRNA,” “guide RNA” and “CRISPR guide sequence” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that associates with the engineered nuclease and determines the sequence specificity of the engineered nuclease. A gRNA may be engineered to hybridize to (e.g., be complementary to, partially or completely) a target nucleic acid sequence (e.g., the genome in a host cell).

[0095] In some embodiments, the at least one gRNA is encoded in a CRISPR RNA (crRNA) array. CRISPR arrays contain a series of direct repeats separated by short sequences called spacers. The engineered nucleases described herein may have a preference for direct repeat sequences. For example, the CRISPR RNA (crRNA) may contain multiple gRNAs or may contain more than one different sequence each configured to hybridize a distinct target nucleic acid sequence.

[0096] The gRNA or portion thereof that hybridizes to the target nucleic acid (a target site) may be between 15-40 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid 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, 59 60, 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).

[0097] In addition to a sequence that binds to a target nucleic acid, 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. |0098] In some embodiments, the gRNA sequence does not comprise a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence.|0099] In some embodiments, the gRNA comprises a sequence of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to a targetnucleic acid. In some embodiments, the sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3’ end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3’ end of the target nucleic acid).

[0100] In some embodiments, the gRNA comprises a spacer sequence. The spacer sequence may be of any length or sequence. In some embodiments, the spacer sequence is at least 18 (e.g., 18, 19, 20, 21, 22, 23, 24, etc.) nucleotides in length. In some embodiments, the spacer sequence is between 18 and 20 nucleotides in length. Thus, in certain embodiments, the spacer sequence is 18 nucleotides in length. In certain embodiments, the spacer sequence is 19 nucleotides in length. In certain embodiments, the spacer sequence is 20 nucleotides in length.

[0101] In some embodiments, the gRNA comprises a spacer sequence complementary to a first strand sequence of the target nucleic acid. In some embodiments, the first strand sequence is directly adjacent to a protospacer adjacent motif (PAM) sequence selected from the group comprising ATTA, GTTA, ATTG, GTTG, TTTA, TTTG, CTTA, CTTC, and CTTG.

[0102] The gRNA may be a non-naturally occurring gRNA.

[0103] In some embodiments, the components of the system may be in the form of a composition. In some embodiments, the components of the present compositions or systems may be mixed, individually or in any combination, with a carrier which are also within the scope of the present disclosure.Exemplary carriers include buffers, antioxidants, preservatives, carbohydrates, surfactants, and the like.

[0104] In some embodiments, the system is a cell free system.

[0105] Also disclosed is a cell comprising the engineered nucleases, fusion proteins, nucleic acids, compositions, or systems 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. In some embodiments, the cell is a human cell.

[0106] In some cases, the compositions or systems disclosed herein may further comprise a donor polynucleotide. For example, in applications in which it is desirable to insert a polynucleotide sequence into the genome where a target sequence is cleaved, a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell. By a “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site targeted by the engineered nuclease (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). In some cases, the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor template is provided to the cell as double-stranded DNA. It may be introduced into acell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor template can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).

[0107] The present disclosure also provides for one or more nucleic acids encoding the engineered nucleases, fusion proteins, and gRNA disclosed herein, vectors containing these nucleic acids and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression cassette in 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.

[0108] In certain embodiments, engineering the nucleases and fusion proteins for use in eukaryotic cells may involve codon- optimization of a nucleic acids encoding the engineered nucleases and fusion proteins. It will be appreciated that changing native codons to those most frequently used in mammals allows for maximum expression of the system proteins in mammalian cells (e.g., human cells). Such modified nucleic acid sequences are commonly described in the art as “codon-optimized,” or as utilizing “mammalian-preferred” or “human-preferred” codons. In some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the codons encoded therein are mammalian preferred codons.

[0109] In some embodiments, the one or more nucleic acids comprise one or more messenger RNAs, one or more vectors, or any combination thereof. In some embodiments, the one or more nucleic acids includes a messenger RNA for expression of the engineered nuclease or fusion protein and at least one nucleic acid provides the gRNA. A single nucleic acid may encode the engineered nuclease or fusion protein and the at least one gRNA, or the engineered nuclease or fusion protein can be encoded on a separate nucleic acid from the at least one gRNA.

[0110] In some embodiments, the engineered nuclease or fusion protein is provided as a split- nuclease or split-protein (e.g., an engineered nuclease can in some cases be delivered as a split-nuclease, or a nucleic acid(s) encoding a split-nuclease) such that two separate proteins together form a functionalnuclease or protein. In some such cases the sequences that encode the two parts of the split-nuclease or split-protein are present on the same vector. In some cases, they are present on separate vectors, e.g., as part of a vector system that encodes the engineered nucleases or fusion protein, the gRNA(s), and systems thereof. Split systems include, but are not limited to, intein, MS2 or SunTag based systems. The split system may comprise more than one split system type (e.g., an intein based system and a SunTag based system) or more than one split system of a single type (e.g., one or more intein based systems).

[0111] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode one or more or all of the components of the present system. 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.

[0112] The vectors of the present disclosure can be delivered to a eukaryotic cell in a subject, such as a mammalian subject, such as a human subject. Modification of the eukaryotic cells via the present system can take place in a cell culture.

[0113] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding components of the present system, or components thereof (e.g., engineered nuclease or fusion proteins), into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding 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), a nucleic acid, and a nucleic acid 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.

[0114] 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 composition or 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.

[0115] A variety of viral constructs can be used to deliver the present composition or system (such as an engineered nuclease, fusion protein, and / or one or more gRNA(s)) to the targeted cells and / or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinantherpes 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( 1): 33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0116] In one embodiment, a DNA segment encoding the engineered nuclease or fusion protein is contained in a plasmid vector that allows expression of the protein and subsequent isolation and purification of the protein produced by the recombinant vector. Accordingly, the engineered nucleases or fusion proteins disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[0117] To construct cells that express the present systems, expression vectors for stable or transient expression of the system, or any of its components, may be constructed via methods as described herein or known in the art and introduced into cells. For example, nucleic acids encoding the 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. In some embodiments, a single nucleic acid comprises a first promoter operatively linked to an engineered nuclease or fusion protein and a second promoter operatively linked to a gRNA. In some cases, the single nucleic acid is a vector.

[0118] In certain embodiments, one or more promoters can drive the expression of one or more sequences (e.g., the engineered nuclease, fusion protein, and / or the gRNA) 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 composition or system may be used with various bacterial hosts.

[0119] In certain embodiments, one or more promoters can drive the expression of one or more sequences (e.g., the engineered nuclease, fusion protein, and / or the gRNA) in mammalian cells, such as when comprised in 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 herein by 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 forboth 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.

[0120] Promoters for use in expressing the engineered nuclease, fusion protein, and / or the gRNA herein may comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue-specific, 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 beta-globin 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. In embodiments, a polymerase II promoter is used to drive expression of the engineered nuclease (e.g., a CMV promoter) and a polymerase III promoter (e.g., U6 promoter) is used to drive expression of the gRNA.

[0121] Different promoters and regulatory elements may be used to achieve proper balance (expression level ratio) between the components of the systems (e.g., the engineered nuclease, the fusion protein, and / or the at least one gRNA). For example, in some cases a nucleic acid includes a promoters and regulatory elements that is operably linked to (and therefore regulates / modulates translation of) a sequence encoding the engineered nuclease or fusion protein. In some cases, a subject nucleic acidincludes a promoters and regulatory elements that is operably linked to a sequence encoding the gRNA. In some cases, the sequence encoding the engineered nuclease or fusion protein and the sequence encoding the gRNA are both operably linked to the same promoters and regulatory elements.

[0122] A variety of promoter types are suitable for use. A promoter can be a constitutively active promoter (e.g., a promoter that is constitutively in an active / ”ON” state), it may be an inducible promoter (e.g., a promoter whose state, active / ”ON” or inactive / ”OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (e.g., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[0123] Moreover, inducible and tissue specific expression of RNA or 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. Promoters 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.

[0124] Examples 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 that 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 engineered nuclease or gRNA operably linked thereto.

[0125] Examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter; a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH); a GnRH promoter; an L7 promoter; a DNMT promoter; an enkephalin; a myelin basic protein (MBP) promoter; a Ca2+-calmodulin-dependent protein kinase II-alpha (CamKIIa) promoter; a CMV enhancer / platelet-derived growth factor-p promoter; and the like. Suitable liver-specific promoters can in some cases include, but are not limited to: TTR, Albumin, and AAT promoters. Suitable CNS- specific promoters can in some cases include, but are not limited to: Synapsin 1, BM88, CHNRB2, GFAP, and CAMK2a promoters. Suitable muscle-specific promoters can in some cases include, but are not limited to: MYODI, MYLK2, SPc5-12 (synthetic), a-MHC, MLC-2, MCK, MHCK7, human cardiac troponin C (cTnC) and desmin promoters. Adipocyte-specific spatially restricted promoters include, but are not limited to, aP2 gene promoter / enhancer, e.g., a region from -5.4 kb to +21 bp of a human aP2; a glucose transporter-4 (GLUT4); a fatty acid translocase (FAT / CD36) promoter; a stearoyl-CoA desaturase- 1 (SCD1) promoter; a leptin promoter; an adiponectin promoter; an adipsin promoter; a resistin promoter; and the like. Cardiomyocyte-specific spatially restricted promoters include, but are not limited to control sequences derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like. Smooth muscle-specific spatially restricted promoters include, but are not limited to, an SM22a promoter; a smoothelin promoter; an a-smooth muscle actin promoter; and the like. For example, a 0.4 kb region of the SM22a promoter, within which lie two CArG elements, has been shown to mediate vascular smooth muscle cell-specific. Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter; a beta phosphodiesterase gene; a retinitis pigmentosagene promoter; an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer; an IRBP gene promoter; and the like.

[0126] Examples of inducible promoters include, but are not limited to, heat shock promoter, tetracycline -regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; an estrogen receptor; an estrogen receptor fusion; an estrogen analog; IPTG; and the like. Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically -regulated promoters such as alcohol-regulated promoters, tetracycline -regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid- regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal- regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0127] Inducible promoters include sugar-inducible promoters (e.g., lactose-inducible promoters; arabinose-inducible promoters); amino acid-inducible promoters; alcohol-inducible promoters; and the like. Suitable promoters include, e.g., lactose-regulated systems (e.g., lactose operon systems, sugar- regulated systems, isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible systems, arabinose regulated systems (e.g., arabinose operon systems, e.g., an ARA operon promoter, pBAD, pARA, portions thereof, combinations thereof and the like), synthetic amino acid regulated systems, fructose repressors, a tac promoter / operator (pTac), tryptophan promoters, PhoA promoters, recA promoters, proU promoters, cst-1 promoters, tetA promoters, cadA promoters, nar promoters, PL promoters, cspA promoters, and the like, or combinations thereof. In certain cases, a promoter comprises a Lac-Z, or portions thereof. In some cases, a promoter comprises a Lac operon, or portions thereof. In some cases, an inducible promoter comprises an ARA operon promoter, or portions thereof. In certain embodiments an inducible promoter comprises an arabinose promoter or portions thereof. An arabinose promoter can be obtained from any suitable bacteria. In some cases, an inducible promoter comprises an arabinoseoperon of E. coli or B. subtilis. In some cases, an inducible promoter is activated by the presence of a sugar or an analog thereof. Non-limiting examples of sugars and sugar analogs include lactose, arabinose (e.g., L-arabinose), glucose, sucrose, fructose, IPTG, and the like. Suitable promoters include a T7 promoter; a pBAD promoter; a lacIQ promoter; and the like. In some cases, the promoter is a J23119 promoter. Many bacterial promoters are known in the art; bacterial promoters can be found on the internet at parts.igem.org / promoters.

[0128] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism is well known in the art. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR)), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems), metal regulated promoters (e.g., metallothionein promoter systems), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter), light regulated promoters, synthetic inducible promoters, and the like.

[0129] Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence capable of driving expression of the desired engineered nuclease, fusion protein, or RNA operably linked thereto.

[0130] Additionally, the vectors described herein for expression of the engineered nucleases, fusion protein, and / or gRNAs 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 oftranscription; 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; S V40 polyoma origins of replication and ColE 1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of 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.

[0131] 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.

[0132] The present compositions and systems (e.g., proteins, polynucleotides encoding these proteins, or compositions comprising the proteins and / or polynucleotides described herein) may be delivered by any suitable means. In certain embodiments, the composition or system is delivered in vivo. In other embodiments, the composition or system is delivered to isolated / cultured cells (e.g., autologous iPS cells) in vitro.

[0133] Vectors and nucleic acids according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of nucleic acid by a host 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.

[0134] Any of the vectors comprising a nucleic acid sequence that encodes the components of the present compositions and system is also within the scope of the present disclosure. Such a vector may bedelivered 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 or RNA, delivery of DNA, RNA, or protein by mechanical deformation, 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.

[0135] 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, biolistics, and the like.

[0136] In some embodiments, the vector is a viral construct, e.g., a recombinant adeno-associated virus construct, a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc. Suitable viral vectors include, but are not limited to, viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex virus; human immunodeficiency virus; a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0137] In some embodiments, the vector is an AAV vector. Adeno-associated virus, or “AAV” encompasses the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise, for example, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV- 10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, ovine AAV, a hybrid AAV (i.e., an AAV comprising a capsid protein of one AAV subtype and genomic material of another subtype), an AAV comprising a mutant AAV capsid protein or a chimeric AAV capsid (i.e. a capsid protein with regions or domains or individual amino acids that are derived from two or more different serotypes of AAV, e.g. AAV-DJ, AAV-LK3, AAV-LK19). “PrimateAAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

[0138] By a “recombinant AAV vector” or “rAAV vector” it is meant an AAV virus or AAV viral chromosomal material comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV), typically a nucleic acid sequence of interest to be integrated into the cell following the subject methods. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). In some instances, the recombinant viral vector also comprises viral genes important for the packaging of the recombinant viral vector material. Packaging refers to the series of intracellular events that result in the assembly and encapsulation of a viral particle, e.g., an AAV viral particle. Examples of nucleic acid sequences important for AAV packaging include the AAV “rep” and “cap” genes, which encode for replication and encapsulation proteins of adeno-associated virus, respectively. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0139] A “viral particle” refers to a single unit of virus comprising a capsid encapsulating a virusbased polynucleotide, e.g., the viral genome (as in a wild-type virus), or, e.g., the subject targeting vector (as in a recombinant virus). An AAV viral particle refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsulated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector.” Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.

[0140] A rAAV virion can be constructed in a variety of methods. For example, the heterologous sequence(s) can be directly inserted into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. In order to produce rAAV virions, an AAV expression vector can be introduced into a suitable host cell using known techniques, such as by transfection. Particularly suitable transfection methods include calcium phosphate co-, direct micro-injection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles.Suitable cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule.

[0141] An AAV virus that is produced may be replication competent or replication-incompetent. A “replication-competent” virus (e.g., a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of being replicated in an infected cell (e.g., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector.

[0142] Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog, and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).

[0143] As noted elsewhere herein, proteins may instead be provided to cells as RNA (e.g., an RNA comprising the translational control element as discussed elsewhere herein). Methods of introducing RNA into cells may include, for example, direct injection, transfection, or any other method used for the introduction of DNA. The engineered nuclease or fusion protein may also be introduced into a host cell directly as protein. In such instances, the engineered nuclease or fusion protein may be delivered as an RNP (ribonucleoprotein complex) in which it is already complexed with an appropriate guide RNA.|0144] The disclosed nucleic acids (e.g., vectors) and proteins can be delivered to cells using any convenient method. Suitable methods include, e.g., viral infection (e.g., AAV, adenovirus, lentiviral), transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0145] In some cases, an engineered nuclease or fusion protein is delivered to a cell in a particle, or associated with a particle. In some cases, an engineered nuclease or fusion protein is delivered with a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1,2-dioleoyl- 3-trimethylammonium-propane (DOTAP) or l,2-ditetradecanoyl-sn-glycero-3 -phosphocholine (DMPC) and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particle further comprises cholesterol.

[0146] An engineered nuclease or fusion protein may be delivered using particles or lipid envelopes. For example, a biodegradable core-shell structured nanoparticle with a poly (p-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self-assembling bioadhesive polymers are used; such particles / nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the desired cell, can be used.

[0147] Lipidoid compounds (e.g., as described in U.S. Patent Application Publication No. 2011 / 0293703) are also useful in the delivery of polynucleotides, and can be used to deliver the disclosed engineered nucleases (or RNA or DNA encoding thereof). In one aspect, the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell to form microparticles, nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0148] A poly(beta-amino alcohol) (PBAA) can be used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a targetcell. U.S. Patent Application Publication No. 2013 / 0302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization.

[0149] Sugar-based particles, for example GalNAc, as described in International Patent Publication No. WO2014118272 (incorporated herein by reference in its entirety and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell.

[0150] In some cases, lipid nanoparticles (LNPs) are used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely l,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinoleyloxy-keto-N,N-dimethyl-3- aminopropane (DLinKDMA), and l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2- DMA). Preparation of LNPs and is described in, e.g., Rosin et al. (2011) Molecular Therapy 19: 1286- 2200). The cationic lipids l,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy- 3-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA), (3-o-[2"- (methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3- [(.omega.-methoxy-poly(ethylene glycol)2000) carbamoyl]- 1 ,2-dimyristyloxlpropyl-3 -amine (PEG-C- DOMG) may be used. A nucleic acid may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40: 10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated.

[0151] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell.

[0152] Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).

[0153] Nanoparticles suitable for use in delivering an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., coreshell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure. In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering an engineered nuclease or nucleic acid to a target cell have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. In some cases, nanoparticles suitable for use in delivering an engineered nuclease or nucleic acid to a target cell have a diameter of from 25 nm to 200 nm.

[0154] In some cases, an exosome is used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs.

[0155] In some cases, a liposome is used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Liposomes are spherical vesicle structures composed of a uni- or multi-lamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of naturalphospholipids and lipids such as 1 ,2-distearoryl-sn-glycero-3 -phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.

[0156] A stable nucleic-acid-lipid particle (SNALP) can be used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. The SNALP formulation may contain the lipids 3-N-[(methoxypoly(ethylene glycol) 2000) carbamoyl] -1,2-dimyristyloxy-propylamine (PEG-C-DMA), l,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC) and cholesterol, in a 2:40: 10:48 molar percent ratio. The SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidyl choline (DSPC), Cholesterol and siRNA using a 25: 1 lipid / siRNA ratio and a 48 / 40 / 10 / 2 molar ratio of Cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA. The resulting SNALP liposomes can be about 80-100 nm in size. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and l,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[0157] Other cationic lipids, such as amino lipid 2, 2-dilinoleyl-4-dimethylaminoethyl-[ 1,3] -di oxolane (DLin-KC2-DMA) can be used to deliver an engineered nuclease or fusion protein or nucleic acid to a target cell. A preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidyl choline (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl- 1 -(methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40 / 10 / 40 / 10, respectively, and a FVII siRNA / total lipid ratio of approximately 0.05 (w / w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+-.0.04 (n=56), the particles may be extruded up to three times through 80 nm membranes prior to adding the guide RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50 / 10 / 38.5 / 1.5) which may be further optimized to enhance in vivo activity.

[0158] Lipids may be formulated with an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, Cl 2-200 and colipidsdisteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with an engineered nuclease or fusion protein or nucleic acid using a spontaneous vesicle formation procedure.

[0159] An engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, may be delivered encapsulated in PLGA microspheres such as those further described in US published applications 20130252281, 20130245107, and 20130244279.

[0160] Supercharged proteins can be used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supemegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can facilitate the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.

[0161] Cell Penetrating Peptides (CPPs) can be used to deliver an engineered nuclease or fusion protein, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids.Methods

[0162] The disclosure also provides methods of modifying a target nucleic acid sequence (e.g., DNA or RNA). The phrase “modifying a nucleic acid sequence,” 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. The modifications may comprise one or more of: modification of the target nucleic acid, modulation of transcription from the target nucleic acid, and modification of a polypeptide associated with a target nucleic acid. In select embodiments, modifying the target nucleic acid comprises one or more of nucleic acid binding, nucleic acid cleavage, base editing, transcriptional modification, and histone modulation. The methods comprise contacting a target nucleic acid sequence with an engineered nuclease, fusion protein, nucleic acid, or composition as disclosed herein, a system disclosed herein, or a composition comprising the system.|0163] In one embodiment, the method introduces 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 both strands of a target DNA sequence, such as within the target genomic DNA sequence and / or within the complement of the target sequence.|0164] In one embodiment, the method introduces a point mutation (e.g., converting a base to another base, such as A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, T to G) in the target nucleic acid sequence through base editing. “Base editing” refers to genome editing technology that involves the conversion of a specific nucleic acid base into another at a target site. In certain embodiments, this can be achieved without requiring a single strand or double strand break in the target nucleic acid sequence. In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional or disease-associated gene, e.g., by correcting a polymorphism in a gene as a result of a nucleic acid point mutation, such as genes comprising pathogenic T>C or A>G mutations. Alternatively, in some embodiments, the purpose of the methods provided herein is to abolish or suppress gene function by the introduction of a mutation as a result of a nucleic acid point mutation.

[0165] In some embodiments, contacting a target nucleic acid sequence comprises introducing the composition or system described herein into the cell. As described above the composition or system may be introduced into eukaryotic or prokaryotic cells by methods known in the art.

[0166] The cell may be a prokaryotic cell, a plant cell, an insect cell, a vertebrate cell, an invertebrate cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is an invertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some cases, the cell is ex vivo (e.g., fresh isolate - early passage). In some cases, the cell is in vivo. In some cases, the cell is in culture in vitro (e.g., immortalized cell line).

[0167] Cells may be from established cell lines or they may be primary cells, where “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in culture.|0168] Suitable cells include, but are not limited to: bacterial cell; an archaeal cell; a eukaryotic cell; a cell of a single-cell eukaryotic organism; a plant cell; a protozoa cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell of a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell of a mammal (e.g., a cell of a rodent; a cell of a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.). In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0169] Non-limiting examples of plant cell include cells from: plant crops, fruits, vegetables, grains, soybean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, fems, clubmosses, homworts, liverworts, mosses, dicotyledons, monocotyledons, seaweeds (e.g., kelp), and the like.]0170] Suitable cells include a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g., a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.|0171] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells,31multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

[0172] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0173] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0174] Adult stem cells are resident in differentiated tissue but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0175] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0176] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver, and yolk sac. HSCs are characterized as CD34+and CD3“. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0177] In other embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblastsor glioblasts, e.g., cells committed to become one or more types of neurons and glial cells, respectively. Methods of obtaining NSCs are known in the art.

[0178] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[0179] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A plant cell can be a cell of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, com, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.

[0180] A plant cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Com, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Dumm), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), bmssels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, com stalks, com- sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head fems, field cress, frisee, gai choy (chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, jemsalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (mssian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms,mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental com, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicomia (sea bean), sinqua (angled / ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

[0181] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a sub-order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola, Hymenoptera, Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0182] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.

[0183] In some embodiments, introducing the system into a cell comprises administering the system to a subject. In some embodiments, the subject is human. The administering may comprise in vivo administration. In alternative embodiments, a vector is contacted with a cell in vitro or ex vivo and the treated cell, containing the system, is transplanted into a subject.

[0184] 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.

[0185] 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, microRNA (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.|0186] The disclosed method may modify a target DNA sequence in a host 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).

[0187] In another embodiment, the method of modifying a target sequence can be used to delete a nucleic acid sequence or portion thereof 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-associated trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research.

[0188] In some embodiments, the systems and methods described herein may be used to insert a gene or fragment thereof into a cell. In particular embodiments, the disclosed systems may be used to generate a cell that expresses a recombinant receptor. In some embodiments, the recombinant receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Also provided herein are cells, e.g., a T cell, comprising a recombinant receptor and / or a nucleic acid encoding thereof and a system (e.g., engineered nuclease and at least one gRNA) as described herein.

[0189] In some embodiments, the system and 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.

[0190] 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 systems and methods may facilitate the production of a new generation of genetically modified crops with various improvedagronomic 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 systems and 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.

[0191] The present system and 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.

[0192] The present disclosure provides for a modified plant cell produced by the present system and 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.

[0193] 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.

[0194] 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.|0195] 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 (i.e., in vitro, ex vivo, or in vivo). Transformation methods based upon the soil bacterium Agrobacterium tumefaciens 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.

[0196] 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 of binary 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.

[0197] 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 particlesare accelerated at high speed into an angiosperm tissue using a device such as the BIOLISTIC PD- 1000 (Biorad; Hercules Calif.).

[0198] In one embodiment, the present systems and methods may be adapted to use in plants. In one embodiment, a series of plant-specific RNA-guided Genome Editing vectors (pRGE plasmids) are provided for expression of the present system 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. In one aspect, the vector constructs include a nucleotide sequence comprising a DNA-dependent RNA polymerase III promoter, wherein the promoter is operably linked to a gRNA molecule and a Pol III terminator sequence, and a nucleotide sequence comprising a DNA-dependent RNA polymerase II promoter operably linked to a nucleic acid sequence encoding the engineered nuclease.

[0199] In certain embodiments, the present systems and 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 systems and 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. In some embodiments, the present system is transiently expressed in plant protoplasts. Vectors for transient transformation of plants include, but are not limited to, pRGE3, pRGE6, pRGE31, and pRGE32. In some embodiment, the vector may be optimized for use in a particular plant type or species, such as pStGE3.

[0200] In one embodiment, the present system may be stably integrated into the plant genome, for example via Agrobacterium-mediated transformation. Thereafter, one or more components of the present system (e.g., the transgene) may be removed by genetic cross and segregation, which may lead to the production of non-transgenic, but genetically modified plants or crops. In one embodiment, the vector is optimized for Agrobacterium-mediated transformation. In one embodiment, the vector for stable integration is pRGEB3, pRGEB6, pRGEB31, pRGEB32, or pStGEB3.

[0201] The present system may be used in various bacterial hosts, 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

[0202] The system and 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 systems and 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 themetagenomic 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.

[0203] 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 of bacterial resistance genes may be disrupted in vivo by insertion of a DNA sequence, leading to non-selective resensitization to drug treatment.

[0204] The components of the composition or system may be administered with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the components of the present system 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.

[0205] In some embodiments, an effective amount of the components of the present system or compositions as described herein can be administered. Within the context of the present disclosure, the term “effective amount” refers to that quantity of the components of the system such that modification of the target nucleic acid is achieved.

[0206] The methods described here also provide for treating a disease or condition in a subject. In some embodiments, the systems and methods are used to treat a pathogen or parasite on or in a subject by altering the pathogen or parasite. In some embodiments, the systems and 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 or polygenic 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.

[0207] 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.

[0208] 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.

[0209] A wide range of additional therapies may be used in conjunction with the methods of the present disclosure. The additional therapy may be administration of an additional therapeutic agent or may be an additional therapy not connected to administration of another agent. Such additional therapies include, but are not limited to, surgery, immunotherapy, radiotherapy. The additional therapy may be administered at the same time as the above methods. In some embodiments, the additional therapy may precede or follow the treatment of the disclosed methods by time intervals ranging from hours to months.|0210] In some embodiments, a therapeutically effective amount of a system (e.g., engineered nuclease, base editor, and / or gRNA) or compositions described herein, is administered alone or in combination with a therapeutically effective amount of at least one additional therapeutic agent. In some embodiments, effective combination therapy is achieved with a single composition or pharmacological formulation or with two distinct compositions or formulations, administered at the same time or separated by a time interval. The at least one additional therapeutic agent may comprise any manner of therapeutic, including protein, small molecule, nucleic acids, and the like. For example, exemplary additional therapeutic agents include, but are not limited to, immune modulators, chemotherapeutic agents, a nucleic acid (e.g., mRNA, aptamers, antisense oligonucleotides, ribozyme nucleic acids, interfering RNAs, antigene nucleic acids), decongestants, steroids, analgesics, antimicrobial agents, immunotherapies, or any combination thereof.

[0211] 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. For example, in connection with cancer the term “treat” may mean elimination or reduction of a patient's tumor burden, or a prevention, delay, or inhibition of metastasis, etc.

[0212] The phrase “pharmaceutically acceptable,” as used in connection with compositions and / or cells of 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.|0213] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, 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.|0214] In some cases, desirable delivery systems provide for roughly uniform distribution and have controllable rates of release of their components (e.g., vectors, proteins, nucleic acids) in vivo. A variety of different media are described below that are useful in creating composition delivery systems. It is not intended that any one medium is limiting to the present invention. Note that any medium may be combined with another medium or carrier; for example, in one embodiment a polymer microparticle attached to a compound may be combined with a gel medium. An implantable device can be used to deliver an engineered nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to, for example, a target cell in vivo.

[0215] Carriers or mediums contemplated include materials such as gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2-hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

[0216] In some cases, a carrier / medium can include a microparticle. 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, pseudo-poly(amino acids), polyhydroxybutyrate -related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids - in any combination thereof. |0217] In some cases, a carrier / medium can include a liposome that is capable of attaching and releasing therapeutic agents (e.g., the subject nucleic acids and / or proteins). Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a therapeutic agent between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid-soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable watersoluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phospholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life.

[0218] In some embodiments, a cationic or anionic liposome is used as part of a subject composition or method, or liposomes having neutral lipids can also be used. Cationic liposomes can include negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate. The choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture.

[0219] Any element of any suitable CRISPR / Cas gene editing system known in the art can be employed in the systems and methods described herein, as appropriate. CRISPR / Cas gene editing technology is described in detail in, for example, U.S. Patent Nos. 8,546,553, 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,889,418; 8,895,308; 8,9066,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; 9,115,348; 9,149,049; 9,493,844; 9,567,603; 9,637,739; 9,663,782; 9,404,098; 9,885,026; 9,951,342; 10,087,431; 10,227,610; 10,266,850; 10,601,748; 10,604,771; and 10,760,064; and U.S. Patent Application Publication Nos. US2010 / 0076057; US2014 / 0113376; US2015 / 0050699; US2015 / 0031134; US2014 / 0357530; US2014 / 0349400; US2014 / 0315985; US2014 / 0310830; US2014 / 0310828; US2014 / 0309487; US2014 / 0294773; US2014 / 0287938; US2014 / 0273230;US2014 / 0242699; US2014 / 0242664; US2014 / 0212869; US2014 / 0201857; US2014 / 0199767;US2014 / 0189896; US2014 / 0186919; US2014 / 0186843; and US2014 / 0179770, each incorporated herein by reference.Kits

[0220] Also within the scope of the present disclosure are kits that include the engineered nucleases, fusion proteins, compositions, systems, or components thereof as disclosed herein.

[0221] For example the kits may contain one or more reagents or other components useful, necessary, or sufficient for practicing any of the methods described herein, such as, editing reagents (engineered nuclease, guide RNAs, vectors, compositions, etc.), transfection or administration reagents, negative andpositive control samples (e.g., cells, template DNA), cells, containers housing one or more components (e.g., microcentrifuge tubes, boxes), detectable labels, detection and analysis instruments, software, instructions, and the like.

[0222] The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of administration of the present system or composition 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.

[0223] 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 access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0224] The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit 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.

[0225] 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.

[0226] The kit may further comprise a device for holding or administering the present system or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and / or a syringe.Examples|0227] The following are examples of the present invention and are not to be construed as limiting.Example 1Generation and Screening of Nuclease Variants|0228] The nuclease of SEQ ID NO: 1 was mutagenized using a random mutagenesis kit (Genemorph II, Agilent Technologies). Mutants were selected using the DEAD / ALIVE bacterial screen approach as described by Chen and Zhao (Nucleic Acids Research, Volume 33, Issue 18, 1 October2005, Page el54, incorporated herein by reference in its entirety). The toxin plasmid contained a target with a CTTC PAM sequence to enable selection for variants with activity against that PAM.

[0229] Vector Construction of nuclease and guide RNA vectors

[0230] Single guide RNA (sgRNA) vectors were placed downstream of the U6 promoter with a starting G, and then placed upstream of the spacer sequence for EMXI or FANCF (shown in Table 1 as EMX1 guide and FANCF guide, respectively).Table 1. Guide and NGS primer sequences for CTTC PAM screen experiments

[0231] Codon-optimized nuclease of SEQ ID NO: 1 was synthesized and cloned into the mammalian expression vector under the CMV promoter, pTwist_CMV (Twist Biosciences). The cloned nuclease was placed into the expression vector with a SV40 Nuclear Localization Sequence (NLS) fused to the N- terminal and a nucleoplasmin NLS on their C-terminal, followed by a 3x HA tag.

[0232] Variants of SEQ ID NO: 1 obtained from the mutagenesis were cloned as a pool by digesting out the SEQ ID NO: 1 backbone and replacing it with the variant pool by ligation. Individual variants were picked and sequenced by nanopore plasmid sequencing to confirm their identity. Variants are listed in Table 2.Table 2. Engineered variants of SEQ ID NO: 1Example 2 Editing Activity in Human Cells

[0233] Nuclease variants in the mammalian expression vector described in Example 1 were tested in HEK293T cells through plasmid transfection using Minis Transit X2 reagent. 50,000 cells were plated per well of a 96 well plate and immediately transfected with 100 ng of nuclease expression vector and 100 ng of the corresponding sgRNA vectors from Example 1 for EMX1 and FANCF. Samples were incubated for 72 h and harvested with QuickExtract (Lucigen). About 200 ng of genomic DNA was amplified using KAPA HiFi polymerase and primers specific to the targeted genomic region (as shown in Table 1 of Example 1) with Illumina adapters. 2 uL of this PCR were input to a second PCR using Illumina P7 barcoded primers from New England BioLabs kit E6609S. PCR products were checked on a 2% agarose gel for purity and cleaned via ZYMO kit D4034. Samples were then sequenced on the Illumina MiSeq system, which returned 100,000-400,000 150bp paired-end reads per sample. Editing analysis was performed by CRISPResso2 with the option cleavage_offset 1” (Clement, Kendell, et al. Nature biotechnology 37.3 (2019): 224-226, incorporated herein by reference). The percentage of nucleotide insertion or deletion mutations (indels) around the cut site was calculated for transfected cells without including substitution-only mutations. The indel percentages of transfected cells were divided by the indel percentage of non-transfected cells to calculate fold change in editing. Results of the editing activity are shown in FIG. 1.Example 3 Base Editors

[0234] Adenine base editor constructs were synthesized according to Kim et al. (Nat Chem Biol. 2022 Sep;18(9):1005-1013, incorporated herein by reference) with the nuclease sequences disclosed herein replacing the nuclease (UnCasl2f) sequence. Base editors were cloned into the mammalian expression vector under the CMV promoter, pTwist_CMV (Twist Biosciences). These constructs (SEQ ID Nos: 17-19) have a catalytically inactivated (dead) version of SEQ ID NO: 1 (dl), containing 2amino acid changes D269A and D467A in SEQ ID NO: 1, fused to a Tad domain (SEQ ID NO: 20) on the C terminus followed by a mutated Tad domain and a nuclear localization signal. The dl and both Tad domains are separated by linkers. The mutated Tad domain for dl-ABE-C2 is Tad-2 (SEQ ID NO: 21). The mutated Tad domain for dl-ABE-C3.0 is Tad-3 (SEQ ID NO: 22). The mutated Tad domain for dl-ABE-C3.1 is Tad-4 (SEQ ID NO: 23).

[0235] To assess activity, the base editors were transfected into HEK293T cells, with harvesting and NGS methods performed as described in Example 2. Table 3 shows the guide sequences for the targets and NGS primer sequences used for sequencing. To assess base editing levels, CRISPResso was run in batch mode with -wc - 10 -w 15 — plot_window_size 40 — base_editor_output — conversion nuc from A - -conversion nuc to G. Results are shown in FIG. 2 as a percentage of editing at each position in the sequence covered by the guide.Table 3. Guide and NGS primer sequences for ABE experimentsSequences

[0236] 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. Variations, 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.

[0237] 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 toclarify 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

CLAIMSWhat is claimed is:

1. An engineered nuclease comprising an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity with any one of SEQ ID NOs: 2- 16, wherein the engineered nuclease has one or more amino acid substitutions, deletions and / or additions as compared to the amino acid sequence of SEQ ID NO: 1.2 An engineered nuclease comprising SEQ ID NO: 1 with one or more amino acid substitutions, deletions and / or additions at an amino acid position selected from 3, 18, 40, 41, 90, 122, 141, 148, 155, 165, 184, 251, 253, 283, 304, 312, 363, 367, 379, 380, 398, 462, 463, and 465.3 The engineered nuclease of claim 2, having 2, 3, 4, 5, 6, 7 or more than 7 amino acid substitutions, deletions and / or additions as compared to the amino acid sequence of SEQ ID NO: 1.4 The engineered nuclease of any one of claims 1-3, comprising the amino acid sequence of SEQ ID Os: 2-16.5 The engineered nuclease of any one of claims 1-4, comprising an amino acid substitution, deletion and / or addition at position 141.6 The engineered nuclease of claim 5, wherein the substitution at position 141 is H141Q.7 The engineered nuclease of any one of claims 1-6, comprising an amino acid substitutions, deletions and / or additions at position 184.8 The engineered nuclease of claim 7, wherein the substitution at position 184 is Q184H.9 The engineered nuclease of any one of claims 1-8, comprising amino acid substitutions, deletions and / or additions at (a) position 141, (b) position 379 or 380 and (c) position 462 or 463.10 The engineered nuclease of claim 9, wherein the substitution at position 141 is H141Q, the substitution at positions 379 and 380 is D379G or E380V and the substitution at position 462 or 463 is E462D or G463A.

11. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 90, 141, and 184.

12. The engineered nuclease of claim 11, comprising the substitutions D90V, H141Q, and Q184H.

13. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 141, 165 and 379.

14. The engineered nuclease of claim 13, comprising the substitutions H141Q, N165Y, and D379G.

15. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 141, 148 and 251.

16. The engineered nuclease of claim 15, comprising the substitutions H141Q, Q148H, and V251L.

17. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 141, 148, 251, 380, and 463.

18. The engineered nuclease of claim 17, comprising the substitutions H141Q, Q148H, V251L, E380V, and G463A.

19. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 40, 141, 148, 251, 283, and 312.

20. The engineered nuclease of claim 19, comprising the substitutions N40Y, H141Q, Q148H, V25 IL, 283Y, and R312Q.

21. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 90, 141, 379, and 462.

22. The engineered nuclease of claim 21, comprising the substitutions D90V, H141Q, D379G, and E462D.

23. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 141, 184, 379, and 463.

24. The engineered nuclease of claim 23, comprising the substitutions H141Q, Q184H, D379G, and G463A.

25. The engineered nuclease of any one of claims 1-5, comprising amino acid substitutions, deletions and / or additions at positions 40, 141, 155, 380, and 463.

26. The engineered nuclease of claim 25, comprising the substitutions N40Y, H141Q, S155G, E380V, and G463A.

27. The engineered nuclease of any one of claims 1-26, wherein the engineered nuclease is modified in its protospacer adjacent motifs (PAM) preference as compared to the PAM preference of SEQ ID NO: 1.

28. The engineered nuclease of any one of claims 1-27, wherein the engineered nuclease further contains one or more mutations or deletions that inactivate the cleavage function of the nuclease.

29. The engineered nuclease of claim 28, wherein the engineered nuclease retains the ability to bind to aarget DNA.

30. The engineered nuclease of any one of claims 1-29, further comprising a nucleic acid localization sequence (NLS).

31. A fusion protein comprising the engineered nuclease of any one of claims 1-30 covalently linked to a second protein or protein domain.

32. The fusion protein of claim 31, wherein the second protein or protein domain comprises an adenosine deaminase, a cytidine deaminase, or a functional fragment thereof.

33. The fusion protein of claim 32, wherein the second protein or protein domain comprises one or ore catalytic domains of the adenosine deaminase (TadA domain).

34. The fusion protein of claim 33, wherein the TadA domain comprises any of SEQ ID NOs: 20-23 and / or the fusion protein comprises any of SEQ ID NOs: 17-19.

35. A nucleic acid encoding the engineered nuclease of any one of claims 1-30 or the fusion protein of any one of claims 31-34.

36. The nucleic acid of claim 35, wherein the nucleic acid is codon-optimized for expression of the engineered nuclease or the fusion protein in a eukaryotic cell.

37. The nucleic acid of claim 35, wherein the nucleic acid is codon-optimized for expression of the engineered nuclease or the fusion protein in a mammalian cell.

38. An expression cassette comprising a promoter operatively linked to the nucleic acid of any one of claims 35-37.

39. A vector comprising the nucleic acid of any one of claims 35-37 or the expression cassette of claim 38.

40. A cell comprising the engineered nuclease of any one of claims 1-30, the fusion protein of any one of claims 31-34, the nucleic acid of any one of claims 35-37, the expression cassette of claim 38, or the vector of claim 39.

41. The cell of claim 40, wherein the cell is a eukaryotic cell.

42. The cell of claim 40, wherein the cell is a mammalian cell.

43. The cell of claim 40, wherein the cell is a human cell.

44. A system for modifying a target comprising the engineered nuclease of any one of claims 1-30, the fusion protein of any one of claims 31-34, the nucleic acid of any one of claims 35-37, the expression cassette of claim 38, or the vector of claim 39 and at least one guide RNA (gRNA).

45. The system of claim 44, wherein the gRNA is an engineered gRNA comprising a first region configured to interact with the engineered nuclease and a second region configured to interact with the target.

46. The system of claim 44 or 45, wherein the target comprises a nucleic acid sequence region within a mammalian genome.

47. The system of claim 46, wherein the nucleic acid sequence region is a gene, a regulatory region, an intron, a splice site, or an intergenic region.

48. The system of any one of claims 44-47, wherein the engineered nucleic acid in the presence of the gRNA is capable of modifying the target.

49. The system of claim 48, where modifying the target comprises one or more of nucleic acid binding, nucleic acid cleavage, base editing, transcriptional modification, and histone modulation.

50. The system of any one of claims 44-49, comprising a first nucleic acid comprising the nucleic acid encoding the engineered nuclease or the fusion protein and a second nucleic acid comprising the gRNA or a nucleic acid encoding the gRNA.

51. The system of claim 50, wherein the first nucleic acid and the second nucleic acid are comprised on a single cassette.

52. The system of claim 50, wherein the first nucleic acid and the second nucleic acid are comprised on separate cassettes.

53. The system of any one of claims 50-52, wherein the first nucleic acid is operatively linked to a first promoter and the second nucleic acid is operatively linked to a second promoter.

54. The system of claim 53, wherein the first promoter and / or the second promoter are active in a mammalian cell.

55. The system of claim 53, wherein the first promoter and / or the second promoter are active in a human cell.

56. The system of any one of claims 53-55, wherein the first promoter and the second promoter are the same.

57. The system of any one of claims 53-55, wherein the first promoter and the second promoter are different.

58. The system of any one of claims 50-57, wherein the first nucleic acid and / or the second nucleic acid are comprised on a viral vector.

59. The system of any one of claims 44-58, further comprising one or more cells.

60. The system of any one of claims 44-58, wherein the system is cell-free.

61. A cell comprising the system of any one of claims 44-58.

62. The cell of claim 61, wherein the cell is a prokaryotic cell or a eukaryotic cell.

63. The cell of claim 61, wherein the cell is a mammalian cell.

64. The cell of claim 61, wherein the cell is a human cell.

65. An in vitro assay comprising the system of any one of claims 44-59.

66. A method of modifying a target nucleic acid sequence comprising contacting the target nucleic acid with a targeting composition comprising the engineered nuclease of any one of claims 1-30, the fusion protein of any one of claims 31-33, the nucleic acid of any one of claims 34-37, the expression cassette of claim 38, the vector of claim 39, or the system of any one of claims 44-59.

67. The method of claim 66, wherein the target nucleic acid sequence is in a cell.

68. The method of claim 67, wherein the cell is a prokaryotic or eukaryotic cell.

69. The method of claim 67, wherein the cell is a human cell.

70. The method of any of claims 66-69, wherein contacting a target nucleic acid sequence comprises introducing the targeting composition into the cell.

71. The method of claim 66, wherein contacting a target nucleic acid sequence comprises administering the targeting composition to a subject.

72. The method of any of claims 66-71, wherein the target nucleic acid sequence encodes a gene product.

73. The method of any of claims 66-72, wherein the targeting composition comprises at least one non- naturally occurring gRNA.

74. A method for treating a disease or disorder in a subject comprising administering to the subject in need thereof a targeting composition comprising the engineered nuclease of any one of claims 1-30, the fusion protein of any one of claims 31-33, the nucleic acid of any one of claims 34-37, the expression cassette of claim 38, the vector of claim 39, the cell of any one of claims 40-43, or the system of any one of claims 44-59 and at least one guide RNA (gRNA) complementary to at least a portion of a target nucleic acid or a nucleic acid comprising a sequence encoding the at least one gRNA.

75. The method of claim 74, wherein the target nucleic acid is a disease-associated gene.

76. The method of claim 74 or claim 75, wherein the subject is a human.

77. Use of the engineered nuclease of any one of claims 1-30, the fusion protein of any one of claims 31-33, the nucleic acid of any one of claims 34-37, the expression cassette of claim 38, the vector of claim 39, the cell of any one of claims 40-43, or the system of any one of claims 44-59 for modifying a target nucleic acid sequence.

78. Use of the engineered nuclease of any one of claims 1-30, the fusion protein of any one of claims 31-33, the nucleic acid of any one of claims 34-37, the expression cassette of claim 38, the vector of claim 39, the cell of any one of claims 40-43, or the system of any one of claims 44-59 for treating a disease or disorder in a subject.

Images