WO2023173062A2 - Nucleic acid editing systems, methods, and uses thereof - Google Patents

Nucleic acid editing systems, methods, and uses thereof Download PDF

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WO2023173062A2
WO2023173062A2 PCT/US2023/064097 US2023064097W WO2023173062A2 WO 2023173062 A2 WO2023173062 A2 WO 2023173062A2 US 2023064097 W US2023064097 W US 2023064097W WO 2023173062 A2 WO2023173062 A2 WO 2023173062A2
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nucleic acid
seq
nuclease
sequence
amino acid
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French (fr)
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WO2023173062A3 (en
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Tilmann BUERCKSTUEMMER
Henrik Schmidt
Anna SIEBER
Christoph ILSINGER
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Intima Bioscience, Inc.
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Publication of WO2023173062A2 publication Critical patent/WO2023173062A2/en
Publication of WO2023173062A3 publication Critical patent/WO2023173062A3/en

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • nucleic acid editing systems will advance gene editing capabilities in biotechnology, medical applications, and synthetic biology.
  • BRIEF SUMMARY The compositions, methods, and kits provided herein are based, in part, on the discovery of new nuclease proteins and nucleic acid editing systems for use in selectively modifying gene expression and gene editing applications.
  • nucleases Provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof.
  • nucleases wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof.
  • nucleases comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof.
  • a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 1.
  • a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 2.
  • a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 10.
  • a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 65. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 67. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 69. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 73.
  • a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 75. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 78. In some embodiments, a nuclease provided herein is encoded by a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90.
  • nucleases comprising: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 15, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe.
  • NUC nuclease
  • REC helical recognition
  • nucleases comprising: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe.
  • NUC nuclease
  • REC helical recognition
  • nucleases comprising: (a) a nuclease (NUC) lobe comprising a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 20; and (b) a helical recognition (REC) lobe.
  • NUC nuclease
  • PAM Protospacer Adjacent Motif
  • PI Protospacer Adjacent Motif
  • REC helical recognition
  • nucleases comprising: (a) a nuclease (NUC) lobe comprising a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 21; and (b) a helical recognition (REC) lobe.
  • NUC nuclease
  • PAM Protospacer Adjacent Motif
  • PI Protospacer Adjacent Motif
  • REC helical recognition
  • nucleases comprising: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe.
  • NUC nuclease
  • WED2 wedge 2
  • nucleic acid sequences wherein the nucleic acid sequences encode for a nuclease provided herein.
  • ribonucleoprotein (RNP) complexes wherein the RNP complexes comprises: (i) a nuclease provided herein; and (ii) a guide RNA that binds to a target nucleic acid.
  • vectors wherein the vectors comprise a nucleic acid sequence encoding for a nuclease provided herein.
  • viral vectors wherein the viral vectors comprise a nucleic acid sequence encoding for a nuclease provided herein.
  • nucleic acid editing systems comprising a nuclease provided herein.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77; a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprising: a nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64; wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease; the guide nucleic acid; and the target nucleic acid form a complex.
  • REC helical recognition
  • nucleic acid editing systems comprising: a nuclease comprising (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64; wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of: SEQ ID NO: 5 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of: SEQ ID NO: 6 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid.
  • compositions wherein the compositions comprise a delivery vehicle; and a nuclease provided herein or a polynucleotide sequence encoding for a nuclease provided herein.
  • compositions wherein the compositions comprise: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof.
  • the nucleic acid comprises a sequence that is at least 85% identical to any one of SEQ ID NOS: 85-90, or 183.
  • viral vectors comprising: a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof.
  • viral vectors comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid.
  • viral vectors wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NO: 5 or SEQ ID NO: 6.
  • kits comprising a nuclease provided herein.
  • kits comprising: a first container comprising a nuclease provided herein; and a second container comprising a guide nucleic acid provided herein, packaging and materials therefor. Further provided herein are kits, wherein the kits comprise: a gene editing system provided herein, packaging and materials therefor.
  • a target nucleic acid molecule comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182; and (b) a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex, wherein the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule.
  • methods of ex vivo modifying a cell comprising: contacting a cell with a composition provided herein under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell.
  • the methods comprise: (a) immobilizing a guide nucleic acid onto a solid support; and (b) contacting the guide nucleic acid with: (i) a test sample; and (ii) a nuclease comprising an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182; wherein when the test sample comprises a target nucleic acid capable of binding to the guide nucleic acid, a complex is formed between the guide nucleic acid, the nuclease, and the target nucleic acid, and wherein upon formation of the complex the nuclease cleaves the target nucleic acid; and (c) detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample.
  • nuclease comprises: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable
  • scaffolds comprising a nuclease or a gene editing system provided herein, wherein the nuclease or the gene editing system is immobilized to the scaffold.
  • the scaffolds comprise: (a) a set of nucleases, wherein at least one nuclease comprises an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182, a functional fragment, or a derivative thereof; and (b) a set of guide nucleic acids, wherein the set of nucleases and/or the set of guide nucleic acids are immobilized to the scaffold.
  • systems and devices comprising: (a) a scaffold provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when a target nucleic acid forms a complex with the scaffold, the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector.
  • systems and devices comprising: (a) a nuclease provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when the nuclease cleaves a target nucleic acid the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector.
  • fusion proteins comprising a nuclease provided herein, a fragment, or a derivative thereof.
  • fusion proteins comprising: (a) a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas poly
  • fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving
  • fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182, a functional fragment, or a derivative thereof; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferas
  • fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a first nuclease consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase,
  • isolated proteins wherein the isolated proteins comprise: an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. Further provided herein are isolated proteins, wherein the isolated proteins comprise any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. [0035] Provided herein are isolated nucleic acids, wherein the isolated nucleic acids comprise: a sequence that encodes a protein that comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof.
  • isolated nucleic acids comprising: a sequence that encodes a protein that comprises an amino acid sequence comprising any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof.
  • engineered nucleic acids wherein the engineered nucleic acids comprise: a sequence that is at least 85% identical to SEQ ID NO: 90 or SEQ ID NO: 183.
  • engineered nucleic acids wherein the engineered nucleic acids comprise SEQ ID NO: 90 or SEQ ID NO: 183.
  • FIGURES 1A-1B shows a schematic representation of a gene editing system.
  • FIG. 1A shows a nuclease provided herein, including a NUC lobe, a REC lobe, and associated domains.
  • FIG. 1A shows a nuclease provided herein, including a NUC lobe, a REC lobe, and associated domains.
  • FIG. 1B shows a gRNA provided herein including a direct repeat (DR) sequence that is a scaffold for the nuclease and a variable sequence that is at least partially complementary to a target nucleic acid.
  • FIGURES 2A-2C show nucleic acid editing system targeting of CD274 (also called PD-L1).
  • FIG. 2A shows a schematic representation of the protocol used to determine nuclease targeting in RKO cells.
  • FIG. 2B shows flow cytometry analysis of CD274 expression in cells transfected with DNA encoding Nuclease #1 without a guide RNA (CNTRL Nuc) and Nuclease #1 (SEQ ID NO: 10) with CD274-targeting gRNA, SEQ ID NO: 53.
  • CNTRL Nuc guide RNA
  • SEQ ID NO: 10 Nuclease #1
  • FIGURE 3 shows a T7 endonuclease assay demonstrating indel formation for control cells (not transfected with a nuclease), a CNTRL Nuc, test nucleases, and Nuclease #1 and Nuclease #2 as indicated. Cleavage products are indicated with arrows.
  • FIGURE 4A-4B shows graphs demonstrating of the percentage and identity of indels produced when cells were treated with an CNTRL Nuc-gRNA; Nuclease #2-gRNA; and Nuclease #1-gRNA.
  • FIG. 4A shows indel/mismatch % for nucleases and a CD274-targeting gRNA (SEQ ID NO: 53). Next generation sequencing (NGS) results for Nuclease #1 are shown.
  • FIG. 4B shows indel/mismatch % for nucleases and a B2M-targeting gRNA (SEQ ID NO: 38). Next generation sequencing (NGS) results for Nuclease #1 are also shown.
  • FIG. 4A shows indel/mismatch % for nucleases and a CD274-targeting gRNA (SEQ ID NO: 53). Next generation sequencing (NGS) results for Nuclease #1 are shown.
  • FIG. 4B shows indel/mismatch % for nucle
  • FIGURE 5 shows flow cytometry analysis of RKO cells contacted with Nuclease #1 and guide nucleic acids SEQ ID NOS: 53-56.
  • FIGURE 6 shows flow cytometry analysis of RKO cells contacted with Nuclease #2 and guide nucleic acids SEQ ID NOS: 57-60.
  • FIGURES 7A-7B show a graph of flow cytometry and gel electrophoresis after T7 endonuclease assay for RKO cells transfected with a guide sequence targeting CD274 and a control or mutant nuclease.
  • FIG.7A is a graph showing the percentage of CD247 negative RKO cells for each of the following conditions: (1) CNTRL Nuclease (SEQ ID NO: 1), no guide; (2) CNTRL Nuclease + Guide; (3) REC Mutant # 1 + Guide (E155R, SEQ ID NO: 65); (4) WED2 Mutant #1 + Guide (S532R, SEQ ID NO: 67); (5) WED2 Mutant #2 + Guide (K538R, SEQ ID NO: 69); (6) Double Mutant #1 + Guide (E155R, S532R, SEQ ID NO: 71); (7) Double Mutant #2 + Guide (E155R, K538R, SEQ ID NO: 73); (8) Double Mutant #3 + Guide (S532R, K538R, SEQ ID NO: 75); and (9) Triple Mutant + Guide (E155R, S532R, K538R, SEQ ID NO: 77).
  • FIG. 7B is a T7 endonuclease assay demonstrating indel formation for control cells (not transfected with a nuclease); a CNTRL Nuclease (SEQ ID NO: 1), no guide; CNTRL Nuclease + Guide; REC Mutant # 1 + Guide (E155R, SEQ ID NO: 65); WED2 Mutant #1 + Guide (S532R, SEQ ID NO: 67); WED2 Mutant #2 + Guide (K538R, SEQ ID NO: 69); Double Mutant #1 + Guide (E155R, S532R, SEQ ID NO: 71); Double Mutant #2 + Guide (E155R, K538R, SEQ ID NO: 73); Double Mutant #3 + Guide (S532R, K538R, SEQ ID NO: 75); and Triple Mutant + Guide (E155R, S532R, K538R, SEQ ID NO: 77).
  • a CNTRL Nuclease SEQ ID NO
  • FIGURE 8 shows flow cytometry results for RKO cells transfected with a guide sequence targeting CD46 and a control or mutant nuclease.
  • the percentage of CD46 negative RKO cells is shown for each of the following conditions: (1) CNTRL Nuclease (SEQ ID NO: 1), no guide; (2) CNTRL Nuclease (SEQ ID NO: 1) + Guide; (3) REC Mutant # 1 (SEQ ID NO: 65) + Guide (E155R); (4) WED2 Mutant #1 (SEQ ID NO: 67) + Guide (S532R); (5) WED2 Mutant #2 (SEQ ID NO: 69) + Guide (K538R); (6) Double Mutant #1 (SEQ ID NO: 71) + Guide (E155R, S532R); (7) Double Mutant # 2 (SEQ ID NO: 73) + Guide (E155R, K538R); (8) Double Mutant #3 (SEQ ID NO: 75) + Guide (S532R, K538R); and (9) Triple
  • FIGURE 9 shows flow cytometry results for RKO cells transfected with a guide sequence targeting B2M and a control or mutant nuclease.
  • the percentage of B2M negative RKO cells is shown for each of the following conditions: 1) CNTRL Nuclease (SEQ ID NO: 1), no guide; (2) CNTRL Nuclease (SEQ ID NO: 1) + Guide; (3) REC Mutant # 1 (SEQ ID NO: 65) + Guide (E155R); (4) WED2 Mutant #1 (SEQ ID NO: 67) + Guide (S532R); (5) WED2 Mutant #2 (SEQ ID NO: 69) + Guide (K538R); (6) Double Mutant #1 (SEQ ID NO: 71) + Guide (E155R, S532R); (7) Double Mutant # 2 (SEQ ID NO: 73) + Guide (E155R, K538R); (8) Double Mutant #3 (SEQ ID NO: 75) + Guide (S532R, K538R); and (
  • FIGURES 10A-10C show a schematic and assay results for various nuclear localization sequence configurations for the control nuclease.
  • FIG. 10A shows a schematic of Configuration 1 (SEQ ID NO: 85), Configuration A (SEQ ID NO: 86), Configuration B (SEQ ID NO: 87), and Configuration C (SEQ ID NO: 88).
  • FIG.10B shows the percentage of CD247 negative RKO cells when cells were contacted with a Configuration 1 construct, Configuration A construct, a Configuration B construct, or a Configuration C construct.
  • FIG. 10C shows flow cytometry graphs for each sequence configuration.
  • FIGURE 12 shows the NGS results of RKO cells co-transfected with the WT (SEQ ID NO: 85) or Triple Mutant nucleases (SEQ ID NOS: 89 and 90) and sgRNAs targeting either B2M, CD274 or CD46.
  • Genomic DNA was extracted from transfected RKO cells and genomic loci of B2M, CD274 and CD46 genes were PCR amplified. PCR amplicons of 200 bp, 210 bp or 233 bp were sequenced via MiSeq PE150 paired-end reads.
  • FIGURES 13A-13B show the effect of various PAM sequences on nuclease targeting of CD274.
  • FIG. 13A show a graph of the % of CD247- negative RKO cells treated with the control nuclease and guide sequences targeting CD247.
  • FIGURES 14A-14B show graphs demonstrating Triple Mutant nuclease fitness effects relative to the starting point on day 0.
  • FIG.14A shows a graph of fitness effects assessed on day 7 relative to day 0 (guide RNA library representation)
  • FIG.14B shows a graph of fitness effects on day 21 (final time point) post guide RNA transduction.
  • the dropout effects of the fitness screen are shown as log fold-change (LFC) values relative to day 0 (X-axis).
  • PAM sites e.g., “CTTC”, “TTTC” and “TTCC” are shown on the Y-axis.
  • compositions, kits, methods, and uses thereof for editing a target gene are provided herein.
  • nucleases (2) guide nucleic acids; (3) target nucleic acids; (4) fusion proteins; (5) nuclease activity and efficiency; (6) delivery systems and vectors; (7) pharmaceutical compositions, dosing, and administration; (8) gene editing applications and diagnostics; (9) scaffolds and systems; and (10) kits.
  • Definitions [0055] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. [0056] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
  • the term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value.
  • the amount “about 10” includes amounts from 9 to 11.
  • the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • the term “cell” or “engineered cell” and their grammatical equivalents as used herein refers to a cell of human or non-human origin.
  • fusion polypeptides are used for targeted single-stranded RNA or single-stranded DNA cleavage. In some embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage. In some cases, cleavage refers to processing of an array of guide nucleic acids, such that individual guide nucleic acids are created. Such processing can involve recognition of a direct repeat region of a guide sequence and cleavage upstream of the direct repeat region. [0064]
  • delivery vehicle refers to a formulation when in combination with a composition provided herein (e.g., a nuclease) delivers the composition to a cell.
  • a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions.
  • Methods for determining the function of a nucleic acid include, but are not limited to: genomic Cleavage Detection assays, RT-PCR, protein expression assays, fluorescence in situ hybridization (FISH), reporter splice-correction assays, cellular in vitro assays and animal models.
  • gene editing and its grammatical equivalents as used herein refers to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g., a naturally-existing nuclease or an artificially engineered nuclease) or other methods known to those of ordinary skill in the art.
  • nuclease e.g., a naturally-existing nuclease or an artificially engineered nuclease
  • homolog and its grammatical equivalents as used herein refers to a protein of the same organism which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related.
  • mutants can include the substitution, deletion, and insertion of one or more nucleotides in a polynucleotide. For example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence can be substituted, deleted, and/or inserted.
  • a mutation can affect the coding sequence of a gene or its regulatory sequence.
  • a mutation can also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA and/or the translated polypeptide structure and function [0074]
  • the term “nuclease” and its grammatical equivalents as used herein refers to a naturally occurring or engineered construct or protein that can be guided by a single guide nucleic acid molecule to specifically recognize a target nucleic acid comprising a sequence that binds the guide nucleic acid molecule.
  • a nuclease has nuclease activity or the ability to cleave a phosphodiester bond in a target nucleic acid.
  • partially complementary refers to a nucleic acid sequence (e.g., a variable sequence of a guide nucleic acid) having a substantially complementary sequence to another nucleic acid sequence (e.g., a target nucleic acid sequence) but that differs from the other nucleic acid by at least two or more nucleotides.
  • a partially complementary nucleic acid specifically excludes a population containing sequences that are exactly complementary, that is, a complementary sequence that has 100% complementarity. Therefore, each member of such a partially complementary nucleic acid population differs from other members of the population by two or more nucleotides, including both strands.
  • nucleases [0077] Provided herein are nucleases, nucleic acid editing systems, and methods for use in modifying or altering a target nucleic acid sequence.
  • the nucleases provided herein can be used in various therapeutic, diagnostic, and biotechnological applications as described herein.
  • the nucleic acid editing systems provided herein have high specificity for a target nucleic acid, high efficacy, and high safety.
  • the nucleases provided herein are guided by a single guide nucleic acid (e.g., a guide RNA) that binds downstream of a protospacer adjacent motif (PAM). Binding is mediated by full or partial complementarity of the variable region of the guide RNA.
  • a single guide nucleic acid e.g., a guide RNA
  • PAM protospacer adjacent motif
  • a PAM is a short nucleic acid sequence (usually 2-6 base pairs in length) that precedes the region targeted for cleavage by the nuclease provided herein.
  • the nucleases provided herein specifically cut the target nucleic acid (e.g., DNA) at the distal end of the PAM, by introducing staggered cuts.
  • nucleases that form a complex with a guide nucleic acid and a target nucleic acid provided herein. Further provided herein is a nuclease with DNA cleavage activity. [0079] The nucleases provided herein comprise a helical recognition (REC) lobe that recognizes the target nucleic acid and a nuclease (NUC) lobe, which cuts the target nucleic acid.
  • REC helical recognition
  • NUC nuclease
  • the NUC lobe comprises a wedge 1 (WED 1) domain; a WED 2 domain; a Protospacer Adjacent Motif (PAM) Interacting (PI) domain; a WED 3 domain; a RuvC1 domain; a bridge helix; a RuvC2 domain; a nuclease (Nuc); and a RuvC3 domain.
  • the nuclease comprises a REC lobe comprising a REC1 domain and a REC2 domain.
  • the RuvC domain of the nucleases provided herein comprise three split RuvC motifs: RuvC I, RuvC II, and RuvC III.
  • a nuclease provided herein comprises an amino acid sequence selected from Table 1. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NOS: 64-78, or SEQ ID NO: 182.
  • a nuclease provided herein comprises: SEQ ID NO: 12 and SEQ ID NO: 14. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 12 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 14 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 13 and SEQ ID NO: 15.
  • a nuclease provided herein comprises: SEQ ID NO: 13 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 15 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 27. [0082] In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 66 and SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 66 and SEQ ID NO: 26.
  • a nuclease provided herein comprises: SEQ ID NO: 64 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 66, SEQ ID NO: 14, and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 68 and SEQ ID NO: 15. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 68 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 15 and SEQ ID NO: 27.
  • a nuclease provided herein comprises: SEQ ID NO: 68, SEQ ID NO: 15, and SEQ ID NO: 27.
  • Additional combinations of nuclease amino acid sequences can include but are not limited to the sequences provided in Table 2.
  • the amino acid sequences in Table 2 can be combined to generate a functional nuclease provided herein (e.g., a nuclease that cleaves a target nucleic acid).
  • a nuclease provided herein comprises at least two amino acid sequences selected from Table 2, in any combination.
  • Table 2 Nuclease Amino Acid Sequence Combinations.
  • nucleases comprising an amino acid sequence that is derived from one or more bacteria.
  • the nuclease is a chimeric nuclease, wherein the chimeric nuclease comprises an amino acid sequence that is derived from two or more different bacteria.
  • a nuclease provided herein comprises an amino acid sequence that is derived or isolated from a bacterium of the genus Prevotella.
  • a nuclease provided herein comprises an amino acid sequence that is derived from a Prevotella ruminicola bacterium.
  • a nuclease provided herein comprises an amino acid sequence that is derived from a P. bryantii bacterium, a P. brevis bacterium, or a P. albensis bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Prevotella ruminicola, wherein the Prevotella ruminicola is of the strain ATCC 19189 / JCM 8958 / 23. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived or isolated from a bacterium of the genus Rumminococcus.
  • a nuclease provided herein comprises an amino acid sequence that is derived from a Rumminococcus bovis bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Rumminococcus bovis, wherein the Rumminococcus bovis is of the strain ATCC 10801. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus sp. AF37-3AC bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus bromii bacterium.
  • a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus sp. AM36-18. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus sp. AM28-29LB. [0085] In some embodiments, a nuclease provided herein further comprises a nuclear localization sequence (NLS). In some embodiments, the nuclease comprises more than one nuclear localization sequence (NLS). An NLS targets a protein to the nucleus of a cell, localizing a nuclease provided herein in close proximity to a target nucleic acid within the nucleus of a cell.
  • the NLS comprises an amino acid sequence that is at least 99% identical to an NLS sequence listed in Table 3, a functional fragment, or a derivative thereof. In some embodiments, the NLS comprises an amino acid sequence that is 100% identical to an NLS sequence listed in Table 3, a functional fragment, or a derivative thereof. Table 3. Nuclear Localization Sequences.
  • a nuclease provided herein comprises an SV40 NLS. In some embodiments, a nuclease provided herein comprises a nucleoplasmin nuclear localization sequence. In some embodiments, a nuclease provided herein comprises an SV40 NLS and a nucleoplasmin nuclear localization sequence.
  • a nuclease provided herein comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 63, or SEQ ID NO: 153.
  • a nuclease provided herein comprises a linker.
  • a linker is a molecular entity that can directly or indirectly connect at two parts of a composition, e.g., from the first protein construct to the second protein construct and the second protein construct to a third protein construct, and so on. Linkers can be configured according to a specific need, e.g., stability or length between two amino acid sequences.
  • linkers can be configured to allow multimerization of at least two nucleases provided herein (e.g., to from a di-, tri-, tetra-, penta-, or higher multimeric complex) while retaining biological activity (e.g., cleavage of a target nucleic acid or set of target nucleic acids).
  • linkers can be configured to facilitate expression and purification of the nuclease provided herein.
  • a linker can be configured to have any length in a form of a peptide, peptidomimetic, a protein, a nucleic acid (e.g., DNA or RNA), or any combinations thereof.
  • the linker can vary from about 2 to about 4 amino acids long, from about 2 to about 6 amino acids long, from about 2 to about 10 amino acids long, or from about 2 to about 100 amino acids long. Longer or shorter linker sequences can be also used for the nucleases and fusion proteins provided herein. [0089] In some embodiments, the linker can be configured to have a sequence comprising at least one of the amino acids selected from the group consisting of glycine (Gly), serine (Ser), asparagine (Asn), threonine (Thr), methionine (Met) or alanine (Ala).
  • uncharged polar amino acids e.g., Gln, Cys, or Tyr
  • nonpolar amino acids e.g., Val, Leu, Pro, Phe, and Trp
  • the linkers can be of any shape.
  • a linker can be linear.
  • a linker can be folded.
  • a linker can be branched.
  • a linker further comprises a detectable label.
  • a linker can be a chemical linker of any length.
  • chemical linkers can comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NH, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms, such as substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted C 5 -C 12 heteroaryl, substituted or unsubstituted C 5 -C 12 heterocyclyl, substituted or unsubstituted C 3 -C 12 cycloalkyl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, NH, or C(O).
  • the chemical linker can be a polymer chain (branched or linear).
  • the linker is a GSGSGS linker (SEQ ID NO: 52).
  • a linker can comprise non-charged or charged amino acids.
  • a linker can comprise alpha-helical domains.
  • a linker can comprise a chemical cross linker.
  • a linker can be of different lengths to adjust the function of fused domains and their physical proximity.
  • a linker can comprise peptides with ligand-inducible conformational changes.
  • a nuclease provided herein further comprises a tag.
  • the tag is a C-terminal tag.
  • the tag is an N-terminal tag.
  • the tag provided herein can be used to detect the nuclease in a sample or immobilize the nuclease to a solid support (e.g, a column).
  • the tag is a c-myc tag.
  • the tag is a recombinant protein.
  • Non-limiting examples of protein tags include: His tags (e.g., HHHHHH (SEQ ID NO: 155)), chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag (WSHPQFEK) (SEQ ID NO: 156), and glutathione-S-transferase (GST).
  • the tag is a solubilization tag (e.g., thioredoxin (TRX), polyNANP, MBP, and GST).
  • the tag is a FLAG-tag (DYKDDDDK (SEQ ID NO: 157)).
  • the tag is an epitope tag.
  • Non-limiting examples of epitope tags include: an ALFA-tag (SRLEEELRRRLTE (SEQ ID NO: 158)), a V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 159)), a Myc-tag (EQKLISEEDL (SEQ ID NO: 30)), an HA-tag (YPYDVPDYA (SEQ ID NO: 160)), a Spot-tag (PDRVRAVSHWSS (SEQ ID NO: 161)), a T7-tag (MASMTGGQQMG (SEQ ID NO: 162)) and a NE-tag (TKENPRSNQEESYDDNES (SEQ ID NO: 163)).
  • the tag is a HiBiT- tag.
  • the tag is an Fc tag, a Nus-tag, a Biotin Carboxyl Carrier Protein (BCCP) tag, an SPB tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 164)), a Ty tag (EVHTNQDPLD (SEQ ID NO: 165)), an Xpress tag (DLYDDDDK (SEQ ID NO: 166)).
  • the tag is a horseradish peroxidase (HRP), a chloramphenicol acetyltransferase (CAT) beta-galactosidase, a beta-glucuronidase, or a luciferase.
  • the tag is a fluorescent tag or a fluorophore.
  • fluorescent tags include: red fluorescent protein, cjBlue, aeBlue, amilGFP, amilCP, Green fluorescent protein (GFP), mCherry, mOrange, blue fluorescent protein (BFP), yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), Venus, Cerulean, HcRed, DsRed, among many others. Any tag that facilitates the detection or immobilization of a nuclease provided herein or a guide nucleic acid provided herein can be used.
  • a nuclease provided herein comprises at least one amino acid substitution or deletion.
  • a nuclease provided herein comprises at least one amino acid substitution or deletion within the nuclease domain of the nuclease amino acid sequence.
  • the amino acid substitution or deletion is between position 1067 and 1262 of the nuclease amino acid sequence.
  • the amino acid substitution or deletion occurs at position 1165 or position 1186.
  • the amino acid substitution is an R1165A substitution.
  • the amino acid substitution is R1186A substitution.
  • a nuclease is modified to become a catalytically dead nuclease or a partially dead nuclease.
  • a nuclease comprises SEQ ID NO: 61 or SEQ ID NO: 62, a functional fragment, or a derivative thereof.
  • the amino acid substitution is within the WED2 domain of a nuclease provided herein.
  • the amino acid substitution is within the REC1 domain of a nuclease provided herein.
  • a nuclease provided herein comprises one or more amino acid substitution.
  • a nuclease provided herein comprises two or more amino acid substitutions.
  • a nuclease provided herein comprises three or more amino acid substitutions.
  • a nuclease provided herein comprises an amino acid substitution at glutamate 155 (Glu155, E155), serine 532 (Ser 532, S532), lysine 538 (Lys 538, K538), or any combination thereof as compared to the sequence of SEQ ID NO: 1.
  • a nuclease provided herein comprises an amino acid substitution at E155, S532, and/or K538, wherein the amino acid substitution at any one of positions E155, S532, and/or K538 comprises a substitution of an amino acid residue to a different amino acid residue comprising a hydrophobic amino acid residue, a hydrophilic amino acid residue, a charged amino acid residue that is a basic amino acid residue, or an acidic amino acid residue, or an aliphatic amino acid residue.
  • the different amino acid residue comprises a charged amino acid residue that is a basic amino acid residue.
  • the different amino acid residue is an arginine (Arg, R) or a lysine (Lys, K), a glutamate (Glu, E), an aspartate (Asp, D), or a valine (Val, V).
  • a nuclease provided herein comprises an amino acid substitution listed in Table 8.
  • a nuclease provided herein comprises an amino acid sequence of any one of SEQ ID NOS: 64-78.
  • a nuclease provided herein is encoded by any one of the nucleic acid sequences of SEQ ID NOS: 85-90, or 183.
  • a nuclease provided herein is encoded by a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90, or 183.
  • Methods of modifying a nucleic acid sequence encoding a nuclease include, e.g., direct mutagenesis or random mutagenesis.
  • Guide Nucleic Acids [0097] Provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise a guide nucleic acid.
  • a nuclease provided herein binds to a guide nucleic acid provided herein.
  • a guide nucleic acid is a guide DNA (gDNA) or a guide RNA (gRNA).
  • the guide nucleic acid comprises RNA.
  • the guide nucleic acid comprises RNA and/or DNA.
  • the guide nucleic acid comprises RNA and a non- canonical nucleobase or non-canonical nucleoside.
  • Canonical nucleobases include, for example, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).
  • Nucleoside analogues are non-canonical nucleosides which contain a nucleobase analogue and a sugar.
  • nucleosides transition to nucleotides capable of incorporation into growing RNA strands. While non-canonical nucleosides are capable of incorporating into a newly synthesized nucleic acid strand, they are generally not capable of being “read” by an RNA polymerase for amplification and act as chain terminators that stop an RNA polymerase.
  • Modified nucleobases which can be incorporated into modified nucleosides and nucleotides and be present in the guide nucleic acid include: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6- methyladenosine), s2U (2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl)
  • any one or any combination of these modified nucleobases may be included in the guide sequence. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the guide nucleic acid can contain phosphoramidate, phosphorothioate, and/or methylphosphonate linkages. [0099] Guide nucleic acids provided herein that comprise at least one modified nucleotide can be prepared using any suitable method. Several suitable methods are known in the art for producing nucleic acid molecules that contain modified nucleotides.
  • the guide nucleic acid is from about 1 base pair to about 30 base pairs in length. In some embodiments, the guide nucleic acid is at least partially complementary to a target nucleic acid sequence. In some embodiments, the guide nucleic acid binds a target nucleic acid. In some embodiments, the guide nucleic acid is at least about 75% complementary to a target polynucleotide sequence.
  • the guide nucleic acid is at least about 80% , 85%, 90%, 95%, 99% complementary to a target nucleic acid sequence. In some embodiments, a guide nucleic acid is fully or 100% complementary to a target nucleic acid sequence.
  • Guide nucleic acids provided herein can comprise a direct repeat sequence.
  • a direct repeat sequence is generally within 20 to 30 base pairs (bps) in length.
  • the direct repeat sequence is at least about 16 bps in length or more, 17 bps in length or more, 18 bps in length or more, 19 bps in length or more, 20 bps in length or more, 21 bps in length or more,, 22 bps in length or more, 23 bps in length or more, 24 bps in length or more, 25 bps in length or more, 26 bps in length or more, 27 bps in length or more, 28 bps in length or more, 29 bps in length or more, 30 bps in length or more.
  • guide nucleic acids provided herein comprise one or more, two or more, three or more, or four or more direct repeat sequences.
  • the length and sequence composition of the guide nucleic acid provided herein may influence the binding of the nuclease provided herein to the guide nucleic acid and the target nucleic acid.
  • the direct repeat sequence comprises a short stem-loop structure.
  • the direct repeat sequence comprises a sequence of SEQ ID NO: 5 or SEQ ID NO: 6, a functional fragment, or a derivative thereof.
  • the guide nucleic acid comprise SEQ ID NO: 5 or SEQ ID NO: 6; and a sequence that is at least partially complementary to a target nucleic acid sequence. Exemplary guide nucleic acids and their corresponding targets and nucleases are provided in Table 4 below.
  • the guide nucleic acid comprises a sequence that is at least 85% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152.
  • the guide nucleic acid comprises a sequence that is at least 90% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. In some embodiments, the guide nucleic acid comprises a sequence that is at least 95% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. In some embodiments, the guide nucleic acid comprises a sequence that is at least 99% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152.
  • the guide nucleic acid comprises a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152.
  • the guide nucleic acid comprises a variable sequence targeting a nucleic acid encoding for CD274, wherein the variable sequence comprises any one of SEQ ID NOS: 7-9, 36, 79, 113-152.
  • the guide nucleic acid comprises any one of SEQ ID NOS: 53-60, or 113-152.
  • the guide nucleic acid comprises a variable sequence targeting a nucleic acid encoding for B2M, wherein the variable sequence comprises SEQ ID NO: 37 or SEQ ID NO: 81.
  • the guide nucleic acid comprises any one of SEQ ID NOS: 38-39, SEQ ID NO: 84.
  • the guide nucleic acid comprises a variable sequence targeting a nucleic acid encoding for cluster of differentiation 46 (CD46), wherein the variable sequence comprises SEQ ID NO: 80.
  • the guide nucleic acid comprises SEQ ID NO: 83.
  • the guide nucleic acid comprises a sequence of any one of: SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152.
  • nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid.
  • the guide nucleic acid comprises a variable sequence.
  • the variable sequence is at least partially complementary to a target nucleic acid sequence.
  • guide nucleic acids provided herein comprise a direct repeat sequence and a variable sequence.
  • a nucleic acid editing system comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5.
  • a nucleic acid editing system comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5.
  • a nucleic acid editing system comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6.
  • guide nucleic acids provided herein are delivered to a cell via an expression cassette.
  • An expression cassette is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a nucleic acid sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a nucleic acid to be transcribed, operably linked to a promoter.
  • guide nucleic acids provided herein further comprise a promoter sequence.
  • the promotor sequence can be any promoter that permits expression of the guide RNA in a cell.
  • a promoter capable of transcribing the guide RNA in a cell includes a heat shock /heat inducible promoter operably linked to a nucleotide sequence encoding the guide RNA.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • promotors include: a ubiquitin promoter (e.g., U6); a Pol-II promoter; a Pol-III promoter; a Banana Streak Virus Promoter (BSV promoter); a Phospholipid Transfer Protein Promoter (PLTP promoter); H1, EF-1a, and a T7 promoter.
  • guide nucleic acids provided herein comprise a promoter sequence, a direct repeat sequence, and a variable sequence.
  • the guide nucleic acid optionally, comprises a polynucleotide linker. In some embodiments, the guide nucleic acid comprises two direct repeat sequences and a variable sequence, wherein the variable sequence is at least 75% complementary to a target nucleic acid sequence.
  • Guide nucleic acids provided herein may comprise non-naturally occurring sequences or engineered sequences.
  • a guide nucleic acid sequence can comprise synthetic RNA or DNA molecules.
  • guide nucleic acids can be generated by transducing or transfecting cells with a plasmid DNA that encodes for an expression cassette.
  • a U6 promoter can be used to drive the expression of the guide RNA.
  • the U6 promoter comprises the sequence of SEQ ID NO: 31.
  • a guide nucleic acid provided herein can be at least partially complementary to a target polynucleotide sequence of interest.
  • a target nucleic acid provided herein comprises a gene sequence.
  • the gene sequence is a mammalian gene sequence.
  • the gene sequence is a human gene sequence.
  • a target nucleic acid provided herein comprises a DNA.
  • a target nucleic acid provided herein comprises a double-stranded DNA.
  • a target nucleic acid provided herein comprises a single-stranded DNA.
  • a target nucleic acid provided herein comprises RNA.
  • a target nucleic acid provided herein comprises single stranded RNA. In some embodiments, a target nucleic acid provided herein comprises double stranded RNA. In some embodiments, a target nucleic acid binds to a variable sequence of a guide nucleic acid. In some embodiments, the target nucleic acid comprises a protospacer-adjacent motif ("PAM"), wherein the PAM is a short T-rich sequence. In some embodiments, cleavage of a target nucleic acid occurs downstream from the PAM sequence. Molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In some embodiments, the PAM is a T-rich PAM.
  • PAM protospacer-adjacent motif
  • the PAM has the nucleotide sequence (T)XN, wherein the X is the number of thymines (e.g, 1-10), and N is A, G, C or T. In certain embodiments, X is equal to 2, and thus, the PAM is TTN. In some embodiments, X is 3, and thus, the PAM is TTTN.
  • a nuclease provided herein recognizes the sequence motif TTTN and directs cleavage of a target nucleic acid sequence 1-24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) bp downstream from that sequence.
  • the PAM site is a TTTA sequence or a TTTC sequence. In some embodiments, the PAM site is a sequence listed in Table 13.
  • the target nucleic acid sequence can be from any cell or organism. Determining the appropriate sequence for the guide nucleic acid to bind to a target nucleic acid provided herein may depend on the sequence of the target and structure of the nuclease provided herein. Methods of designing a guide nucleic acid for gene editing are known in the art and include, e.g., using software and databases such as Breaking-Cas, Cas-OFFinder, CRISPR-DT, CHOPCHOP, CCTOP, CRISPick, or CRISPOR.
  • the target nucleic acid sequence is a mammalian DNA sequence. In some embodiments, the target nucleic acid sequence is a human DNA sequence. In some embodiments, the target nucleic acid sequence is a sequence expressed by a tumor or a cancer cell. In some embodiments, the target nucleic acid comprises a sequence associated with a disease or condition. In some embodiments, the target nucleic acid sequence is a sequence expressed by an immune cell.
  • Non-limiting examples of target nucleic acids include nucleic acid encoding: cluster of differentiation 274 (CD274, also called programmed cell death ligand 1 or PD-L1), beta-2-microglobulin (B2M), CD46, CD81, C-C chemokine receptor type 5 (CCR5), BAF Chromatin Remodeling Complex Subunit 11 (BCL11A), transthyretin (TTR), programmed cell death 1 (PD-1), programmed cell death 2 (PD-2), T cell immunoglobulin and ITIM domain (TIGIT), Lymphocyte Activating 3 (LAG3), and Cytokine-inducible SH2-containing protein (CISH).
  • CD274 cluster of differentiation 274
  • B2M beta-2-microglobulin
  • CD46 CD81
  • CD81 C-C chemokine receptor type 5
  • CCR5 C-C chemokine receptor type 5
  • BCL11A BAF Chromatin Remodeling Complex Subunit 11
  • TTR transthyretin
  • Fusion proteins comprising a nuclease provided herein.
  • the nucleases provided herein can be fused to one or more additional protein constructs for a particular application (e.g., therapeutic gene editing or cellular gene editing for biotechnology applications in vitro or ex vivo ). Multimerization of an effector or protein tag by direct fusion with the nuclease provided herein can localize the effector domain to a site matched by the guide nucleic acid provided herein.
  • the fusion proteins provided herein can be used to modulate transcription, expression, translation, RNA splicing, methylation status, DNA unwinding, or any other property that alters nucleic acid expression in a cell.
  • nucleases provided herein are fused to at least one additional element.
  • a fusion protein comprises a first protein construct comprising a nuclease provided herein.
  • a fusion protein provided herein comprises a second protein construct.
  • fusion proteins provided herein optionally, comprise a linker between the first protein construct and second protein construct.
  • fusion proteins provided herein further comprise a third, fourth, and/or fifth protein construct.
  • a fusion protein provided herein comprises one or more linkers between one or more protein constructs.
  • a fusion protein provided herein comprises a cleavable or non-cleavable linker between the different protein constructs or domains of the fusion protein.
  • a linker can be a polypeptide linker, such as a linker that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids long.
  • two polypeptide constructs that are “fused” need not be directly adjacent to each other.
  • Fused polypeptide sequences can be fused by a linker, or by an additional functional or non-functional polypeptide sequence that is fused to the polypeptide sequences.
  • the number of additional elements in the nuclease-fusion protein will depend on a number of factors, including, e.g., size or molecule weight of the fusion protein for delivery to a cell, or translocation of the fusion protein desired in a cell type of interest.
  • a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof.
  • a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof. In some embodiments, a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof.
  • a fusion proteins provided herein comprise a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 99% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof.
  • a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is 100% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof.
  • a fusion protein provided herein comprises one or more amino acid sequence selected from Table 1.
  • a fusion protein comprises: a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14; or SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid.
  • REC helical recognition
  • the first protein construct comprises a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid.
  • NUC nuclease
  • WED2 wedge 2
  • REC helical recognition
  • the first protein construct (e.g., a first nuclease) is capable of cleaving the target nucleic acid via the NUC lobe.
  • a fusion protein provided herein comprises a second protein construct.
  • a fusion protein provided herein comprises a protein construct selected from Table 5.
  • Table 5 provides a list of proteins that can be fused to a nuclease provided herein, a functional fragment, or a derivative thereof.
  • Table 5 also provides the function of various proteins and exemplary NCBI/UniProt accession numbers, which can be selected using ordinary skill in the art, for instance, based on amino acid sequence, crystal structure data, computational structure predictions, and functional activity. Table 5. Protein constructs.
  • a fusion protein provided herein comprises a protein construct comprising an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 5, a functional fragment, or a derivative thereof.
  • a fusion protein provided herein comprises a protein construct comprising a protein from Table 5, a homolog, or an ortholog thereof.
  • Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling, e.g., BLAST, NCBI, or UniProt.
  • the additional protein construct is of eukaryotic origin, such as of human, rat or lamprey origin.
  • the additional protein construct is of prokaryotic origin, such as a bacterium or archaeon.
  • the additional protein construct comprises a viral protein.
  • a fusion protein provided herein comprises one or more additional protein constructs, wherein the one or more additional protein constructs comprises: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease,
  • the nickase comprises a sequence that is at least 90% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nickase comprises a sequence that is at least 95% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nickase comprises a sequence comprising SEQ ID NO: 61. In some embodiments, the nickase comprises a sequence comprising SEQ ID NO: 62.
  • the fusion protein provided herein comprises a nuclease deficient protein construct, wherein the nuclease deficient protein construct comprises a sequence that is at least 85% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nuclease deficient protein construct comprises a sequence that is at least 90% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nuclease deficient protein construct comprises a sequence that is at least 95% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nuclease deficient protein construct comprises a sequence comprising SEQ ID NO: 61.
  • the nuclease deficient protein construct comprises a sequence comprising SEQ ID NO: 62.
  • a fusion protein provided herein comprises a protein construct that modulates transcription.
  • a fusion protein provided herein comprises a transcriptional repressor protein construct.
  • a fusion protein provided herein comprises a zinc finger protein construct.
  • the zinc finger protein construct comprises a Krüppel-associated box (KRAB) protein or a functional fragment thereof.
  • the fusion protein comprises a KRAB domain that binds to a transcriptional corepressor protein.
  • a fusion protein provided herein comprises a SUMO protein construct.
  • a fusion protein provided herein comprises a transcriptional activator protein construct.
  • a transcriptional activator protein construct recruits transcription factors from a host cell to the target nucleic acid for regulation of target nucleic acid expression.
  • Exemplary transcriptional activators include VP64, VP16, VP160, VP48, VP96, p65, Rta, VPR, hsf1, and p300.
  • a fusion protein provided herein comprises one or more VP16 protein construct.
  • a fusion protein provided herein comprises one or more VP64 protein construct.
  • a fusion protein provided herein comprises one or more VPR protein construct.
  • a fusion protein provided herein comprises one or more SunTag protein construct.
  • a fusion protein provided herein comprises VP64, p65, and HSF1 (SunTag-p65-heat-shock factor 1 or SPH).
  • a fusion protein provided herein comprises a CREB-binding protein (CPB).
  • CBP can be used to recruit transcriptional machinery and function as a histone acetyltransferase (HAT) that alters chromatin structure.
  • a nuclease or a fusion protein provided herein further comprises an aptamer.
  • the aptamers are bind to one or more MS2 proteins.
  • a fusion protein provided herein comprises a self-cleaving protein sequence.
  • self-cleaving protein sequences include: E2A, P2A and T2A.
  • the fusion protein comprises an antibiotic resistance protein construct or an antibiotic resistance selectable marker.
  • Non-limiting examples of antibiotic resistance proteins and selectable markers include: aminoglycoside acetyltransferase, rifampin ADP-ribosyltransferase, dihydrofolate reductase, multidrug and toxic compound extrusion transporters, antibiotic resistance ATP-binding cassette family F (ARE ABC-F) proteins (e.g., MsrE, Erm, Vga, Lsa, Sal, OptrA), ⁇ -lactamase, blasticidin-S deaminase, penicillin-binding proteins (PBPs), and puromycin-N-acetyltransferase.
  • a fusion protein provided herein comprises a base editor.
  • fusion proteins provided herein further comprise a uracil-DNA glycosylase, a functional fragment, or a derivative thereof.
  • fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a first nuclease consisting of SEQ ID NO: 1, 2, 10, 11, 77-78; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methyla
  • Fusion proteins provided herein can be synthesized using known technologies, for instance, recombination DNA technology where the coding sequences of various portions of the fusion proteins can be linked together at the nucleic acid level. Subsequently a fusion protein can be produced using a host cell. 5. Methods of Determining Gene Editing Efficiency [00131] Provided herein are methods of determining gene editing efficiency and selectivity of a nuclease or fusion protein provided herein for a target nucleic acid sequence.
  • the target nucleic acid is cleaved by a nuclease provided herein upon binding of the guide sequence to the target nucleic acid sequence.
  • the target nucleic acid is a DNA.
  • the nuclease provided herein cleaves the DNA resulting in a double-stranded break (DSB). In some embodiments, the nuclease provided herein cleaves the target DNA via staggered DNA double-stranded break. In some embodiments, the staggered DNA double-stranded break generates a 5' overhang. The 5' overhang can facilitate gene insertion via non-homologous end joining (NHEJ) mechanisms. In some embodiments, the 5’ overhang is at least 3 base pairs (bps) or more, at least 4 bps or more, at least 5 bps or more, or at least 6 bps or more. In some embodiments, the 5’ overhang is about 4 bps to 5 bps in length.
  • the ability of a nuclease to recognize a PAM sequence can be determined by an in vitro selection assay.
  • the activity of a nucleic acid editing system may be assayed using a cell expressing a reporter protein or containing a reporter gene.
  • a reporter protein may be engineered to contain an obstruction, such as a stop codon, a frameshift mutation, a spacer, a linker, or a transcriptional terminator; the nucleic acid editing system may then be used to remove the obstruction and the resultant functional reporter protein may be detected.
  • the obstruction may be designed such that a specific sequence modification is required to restore functionality of the reporter protein.
  • the obstruction may be designed such that any insertion or deletion which results in a frame shift of one or two bases may be sufficient to restore functionality of the reporter protein.
  • reporter proteins include colorimetric enzymes, metabolic enzymes, fluorescent proteins, enzymes and transporters associated with antibiotic resistance, and luminescent enzymes.
  • reporter proteins include ⁇ -galactosidase, Chloramphenicol acetyltransferase, Green fluorescent protein, Red fluorescent protein, luciferase, and renilla.
  • Different detection methods may be used for different reporter proteins.
  • the reporter protein may affect cell viability, cell growth, fluorescence, luminescence, or expression of a detectable product.
  • the reporter protein may be detected using a colorimetric assay.
  • the reporter protein may be a fluorescent protein, and DNA editing may be assayed by measuring the degree of fluorescence in treated cells, or the number of treated cells with at least a threshold level of fluorescence.
  • transcript levels of a reporter gene may be assessed.
  • a reporter gene may be assessed by sequencing.
  • an assay for measuring DNA editing may use a split fluorescence protein system, such as the self-complementing split GFP1-10/11 systems, in which two fragments (G 1-10 and G 11 ) of the GFP protein which can associate by themselves to form a functional GFP signal are linked using a frameshifting linker.
  • Insertions or deletions within the frameshifting linker can restore the frame of the G11 fragment allowing the two fragments to form a functional GFP signal.
  • Integration of an exogenous nucleic acid can be measured using any technique, e.g., integration can be measured by flow cytometry, surveyor nuclease assay, tracking of indels by decomposition (TIDE), junction PCR, or any combination thereof.
  • TIDE indels by decomposition
  • junction PCR junction PCR
  • transgene integration can be measured by PCR.
  • a TIDE analysis can also be performed on engineered cells.
  • Ex vivo cell transfection can also be used for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism).
  • cells are isolated from the subject organism, transfected with a nucleic acid (e.g., gene or cDNA), and re-infused back into the subject organism (e.g., subject).
  • a nucleic acid e.g., gene or cDNA
  • the amount of nuclease-containing modified cells that can be necessary to be therapeutically effective in a subject can vary depending on the viability of the cells, and the efficiency with which the cells have been genetically modified (e.g., the efficiency with which a transgene has been integrated into one or more cells).
  • the product (e.g., multiplication) of the viability of cells post genetic modification and the efficiency of integration of a transgene can correspond to the therapeutic aliquot of cells available for administration to a subject.
  • an increase in the viability of cells post genetic modification can correspond to a decrease in the amount of cells that are necessary for administration to be therapeutically effective in a subject.
  • an increase in the efficiency with which a transgene has been integrated into one or more cells can correspond to a decrease in the amount of cells that are necessary for administration to be therapeutically effective in a subject.
  • determining an amount of cells that are necessary to be therapeutically effective can comprise determining a function corresponding to a change in the viability of cells over time.
  • determining an amount of cells that are necessary to be therapeutically effective can comprise determining a function corresponding to a change in the efficiency with which a transgene can be integrated into one or more cells with respect to time dependent variables (e.g., cell culture time, electroporation time, cell stimulation time).
  • time dependent variables e.g., cell culture time, electroporation time, cell stimulation time.
  • a percent of NHEJ, HDR, or a combination of both can be determined by co-delivering the gene editing molecules, for example a guide nuclease acid and a nuclease or fusion protein provided herein, with a donor nucleic acid template that encodes a promoter-less tag or marker (e.g., GFP) into cells.
  • a duration of time e.g., 72-96 hours
  • flow cytometry can be performed to quantify the total cell number (NTotal), tag-positive cell number and tag/GFP-negative cell number.
  • next-generation sequencing can be performed to identify cells without mutations and with mutations. HDR efficiency and NHEJ efficiency can be calculated from the assay.
  • Additional assays for determining gene editing efficiency of a nucleic acid editing system for cleaving a nucleic acid include but are not limited to: RT-PCR, nucleic acid sequencing, T7 endonuclease 1 (T7E1) mismatch detection assays, tracking of indels by decomposition (TIDE) assays, and indel detection by amplicon analysis (IDAA) assays.
  • T7E1 T7 endonuclease 1
  • TIDE tracking of indels by decomposition
  • IDAA indel detection by amplicon analysis
  • the indel pattern that is induced at the target site of a programmable nuclease may also be determined by PCR-amplifying the respective region and subsequent next generation sequencing.
  • polynucleotides encoding a nuclease provided herein comprises a promoter sequence.
  • Useful promoters for nuclease encoding sequences include, e.g., CMV, EF- 1a, MSCV, PGK, and CAG control promoters.
  • compositions comprising: a nucleic acid encoding for a nuclease provided herein; and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: one or more nucleic acids encoding for a nucleic acid editing system provided herein and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: one or more nucleic acids encoding for a fusion protein provided herein and a delivery vehicle.
  • compositions comprising: a first nucleic acid encoding for a nuclease provided herein; a second nucleic acid encoding for a guide nucleic acid provided herein; and a delivery vehicle.
  • compositions wherein the compositions comprise: a first nucleic acid encoding for a fusion protein provided herein; a second nucleic acid encoding for a guide nucleic acid provided herein; and a delivery vehicle.
  • compositions comprising: a first nucleic acid encoding for a first protein construct provided herein; a second nucleic acid encoding for a second protein construct; a third nucleic acid encoding for a guide nucleic acid provided herein; and a delivery vehicle.
  • compositions comprisin the compositions comprise: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, or 182, a functional fragment, or a derivative thereof.
  • the composition further comprises an exogenous nucleic acid for delivery to a cell.
  • the exogenous nucleic acid is incorporated into a target nucleic acid sequence.
  • the exogenous nucleic acid encodes for a protein construct provided in Table 5.
  • the nucleases, fusion proteins, guide nucleic acids, and nucleic acid editing systems provided herein can be delivered to a target cell by any suitable means.
  • the nucleases and guide nucleic acids provided herein can be admixed with a delivery vehicle that permits delivery of the system to the target nucleic acid sequence.
  • Non-limiting examples of delivery vehicles include an emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, a nanoparticle, a polymer, microcapsules, recombinant cells, cell culture medium, blood, or serum. Specific types of delivery vehicles that can be used in a composition provided herein are further described below. [00139] In some embodiments, the delivery vehicle is a liposome. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs multilamellar vesicles
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Liposomes interact with cells via different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one can operate at the same time.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles can also be used as a delivery vehicle.
  • the delivery vehicle is a phospholipid. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the liposomes form. Physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability.
  • the phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less- ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
  • the delivery vehicle is a nanoparticle.
  • Nanoparticle carriers that specifically target a tissue provided herein may also be used as a pharmaceutically acceptable carrier.
  • the nanoparticle is a gold nanoparticle, a platinum nanoparticle, an iron-oxide nanoparticle, a lipid nanoparticle, a selenium nanoparticle, a tumor- targeting glycol chitosan nanoparticle (CNP), a cathepsin B sensitive nanoparticle, a hyaluronic acid nanoparticle, a paramagnetic nanoparticle, or a polymeric nanoparticle.
  • CNP tumor- targeting glycol chitosan nanoparticle
  • the nucleic acid editing systems provided herein can be delivered using vectors, for example containing sequences encoding one or more of the nucleases or fusion proteins provided herein.
  • a nucleic acid editing system as described herein can be delivered absent a viral vector.
  • Transgenes encoding polynucleotides can be similarly delivered. Any vector systems can be used including, but not limited to, plasmid vectors, viral vectors, and oncolytic viral vectors. Furthermore, any of these vectors can comprise one or more transcription factor, nuclease, and/or transgene. [00143] In some embodiments, vectors provided herein are a viral vector.
  • Exemplary viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adeno- associated viral vectors (AAV), adenoviral vectors, herpes simplex viral vectors, alphaviral vectors, flaviviral vectors, rhabdoviral vectors, measles viral vectors, Newcastle disease viral vectors, poxviral vectors, and picornaviral vectors.
  • AAV adeno- associated viral vectors
  • polynucleotides encoding an engineered protein provided herein are contained in an AAV viral vector.
  • vectors provided herein are an oncolytic viral vector.
  • polynucleotides encoding for a nuclease or a fusion protein provided herein are contained in an lentiviral viral vector.
  • lentiviral vectors wherein the lentiviral vectors comprise a polynucleotide encoding for a nucleic acid editing system provided herein.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding for a nuclease provided herein, a nucleic acid editing system, and/or transgenes in cells (e.g., mammalian cells) and target tissues.
  • Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by AMAXA ® Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc.
  • Lipofection reagents are sold commercially (e.g., TRANSFECTAM ® and LIPOFECTIN ® ). Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV.
  • EDVs EnGeneIC delivery vehicles
  • Vectors including viral and non-viral vectors containing nucleic acids encoding a nucleic editing system provided herein can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA or mRNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. More than one route can be used to administer a particular composition.
  • a vector encoding for an exogenous transgene can be shuttled to a cellular nucleus.
  • a vector can contain a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • An NLS can be from Simian Vacuolating Virus 40.
  • a vector can also be shuttled by a protein or protein complex.
  • a nuclease provided herein can be used as a means to shuttle a minicircle vector.
  • the exogenous transgene comprises one or more NLS.
  • a vector can be pre-complexed with a nuclease provided herein prior to electroporation into a cell.
  • a nuclease that can be used for shuttling can be a nuclease-deficient or catalytically dead protein.
  • a nuclease that can be used for shuttling can be a nuclease-competent protein.
  • a nuclease provided herein can be pre-mixed with a guide nucleic acid and a vector or plasmid encoding an exogenous transgene.
  • a cell can be transfected with a mutant or chimeric adeno-associated viral vector encoding an exogenous transgene and a nucleic acid editing system comprising a nuclease provided herein.
  • An AAV vector concentration can be from 0.5 nanograms to 50 micrograms.
  • nucleic acid e.g., ssDNA, dsDNA, and/or RNA
  • the amount of nucleic acid (e.g., ssDNA, dsDNA, and/or RNA) that can be introduced into the cell by electroporation can be varied to optimize transfection efficiency and/or cell viability. In some embodiments, less than about 100 picograms of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated).
  • dsDNA 1 microgram of dsDNA can be added to each cell sample for electroporation.
  • the amount of nucleic acid (e.g., dsDNA) required for optimal transfection efficiency and/or cell viability can be specific to the cell type.
  • the amount of nucleic acid (e.g., dsDNA) used for each sample can directly correspond to the transfection efficiency and/or cell viability.
  • the transfection efficiency of cells with any of the nucleic acid delivery platforms described herein, for example, nucleofection or electroporation can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%.
  • Viral particles such as AAV, can be used to deliver a viral vector comprising a gene of interest or a transgene into a cell ex vivo or in vivo.
  • a mutated or chimeric adeno-associated viral vector as disclosed herein can be measured as pfu (plaque forming units).
  • the pfu of recombinant virus or mutated or chimeric adeno-associated viral vector of the compositions and methods of the disclosure can be about 10 8 to about 5 ⁇ 10 10 pfu.
  • recombinant viruses of this disclosure are at least about 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 10 , and 5 ⁇ 10 10 pfu.
  • recombinant viruses of this disclosure are at most about 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 10 , and 5 ⁇ 10 10 pfu.
  • a mutated or chimeric adeno-associated viral vector of the disclosure can be measured as vector genomes.
  • recombinant viruses of this disclosure are 1 ⁇ 10 10 to 3 ⁇ 10 12 vector genomes, or 1 ⁇ 10 9 to 3 ⁇ 10 13 vector genomes, or 1 ⁇ 10 8 to 3 ⁇ 10 14 vector genomes, or at least about 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 , 1 ⁇ 10 14 , 1 ⁇ 10 15 , 1 ⁇ 10 16 , 1 ⁇ 10 17 , and 1 ⁇ 10 18 vector genomes, or are 1 ⁇ 10 8 to 3 ⁇ 10 14 vector genomes, or are at most about 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11
  • a mutated or chimeric adeno-associated viral vector of the disclosure can be measured using multiplicity of infection (MOI).
  • MOI can refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic can be delivered.
  • the MOI can be 1 ⁇ 10 6 GC/mL.
  • the MOI can be 1 ⁇ 10 5 GC/mL to 1 ⁇ 10 7 GC/mL.
  • the MOI can be 1 ⁇ 10 4 GC/mL to 1 ⁇ 10 8 GC/mL.
  • recombinant viruses of the disclosure are at least about 1 ⁇ 10 1 GC/mL, 1 ⁇ 10 2 GC/mL, 1 ⁇ 10 3 GC/mL, 1 ⁇ 10 4 GC/mL, 1 ⁇ 10 5 GC/mL, 1 ⁇ 10 6 GC/mL, 1 ⁇ 10 7 GC/mL, 1 ⁇ 10 8 GC/mL, 1 ⁇ 10 9 GC/mL, 1 ⁇ 10 10 GC/mL, 1 ⁇ 10 11 GC/mL, 1 ⁇ 10 12 GC/mL, 1 ⁇ 10 13 GC/mL, 1 ⁇ 10 14 GC/mL, 1 ⁇ 10 15 GC/mL, 1 ⁇ 10 16 GC/mL, 1 ⁇ 10 17 GC/mL, and 1 ⁇ 10 18 GC/mL MOI.
  • a mutated or chimeric adeno-associated viruses of this disclosure are from about 1 ⁇ 10 8 GC/mL to about 3 ⁇ 10 14 GC/mL MOI, or are at most about 1 ⁇ 10 1 GC/mL, 1 ⁇ 10 2 GC/mL, 1 ⁇ 10 3 GC/mL, 1 ⁇ 10 4 GC/mL, 1 ⁇ 10 5 GC/mL, 1 ⁇ 10 6 GC/mL, 1 ⁇ 10 7 GC/mL, 1 ⁇ 10 8 GC/mL, 1 ⁇ 10 9 GC/mL, 1 ⁇ 10 10 GC/mL, 1 ⁇ 10 11 GC/mL, 1 ⁇ 10 12 GC/mL, 1 ⁇ 10 13 GC/mL, 1 ⁇ 10 14 GC/mL, 1 ⁇ 10 15 GC/mL, 1 ⁇ 10 16 GC/mL, 1 ⁇ 10 17 GC/mL, and 1 ⁇ 10 18 GC/mL MOI.
  • a non-viral vector or nucleic acid can be delivered without the use of a mutated or chimeric adeno-associated viral vector and can be measured according to the quantity of nucleic acid.
  • any suitable amount of nucleic acid can be used with the compositions and methods of this disclosure.
  • nucleic acid can be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 200 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 600 ⁇ g, 700 ⁇ g, 800 ⁇ g, 900 ⁇ g, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.
  • nucleic acid can be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 200 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 600 ⁇ g, 700 ⁇ g, 800 ⁇ g, 900 ⁇ g, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.
  • Proteins, vectors, plasmids, and the nucleic acid editing systems provided herein can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • the methods used to construct any embodiment of the compositions provided herein are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques.
  • a nuclease, nucleic acid editing system, fusion protein, or a polynucleotide encoding a nuclease, nucleic acid editing system, fusion protein can be delivered to a cell by electroporation.
  • Electroporation using, for example, the NEON® Transfection System (ThermoFisher Scientific) or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery of nucleic acids and proteins into a cell.
  • a nuclease or fusion protein provided herein can be purified and complexed with a suitable guide nucleic acid for delivery into a cell.
  • Electroporation parameters can be adjusted to optimize delivery efficiency and/or cell viability.
  • Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance).
  • E Field Strength
  • compositions provided herein can be delivered to any suitable cell.
  • suitable cells can include but are not limited to eukaryotic and prokaryotic cells and/or cell lines.
  • a suitable cell can be a human primary cell.
  • a primary cell can be taken directly from living tissue (i.e.
  • a primary cell can be acquired from a variety of sources such as an organ, vasculature, buffy coat, whole blood, apheresis, plasma, bone marrow, tumor, cell-bank, cryopreservation bank, or a blood sample.
  • a primary cell can be a stem cell.
  • a suitable cells that can be edited with a nucleic acid editing system provided herein include but are not limited to: epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B, NK, and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, ery
  • the one or more cells can be pancreatic islet cells and/or cell clusters or the like, including, but not limited to pancreatic ⁇ cells, pancreatic ⁇ cells, pancreatic ⁇ cells, pancreatic F cells (e.g., PP cells), or pancreatic ⁇ cells.
  • the one or more cells can be pancreatic ⁇ cells.
  • the one or more cells can be pancreatic ⁇ cells.
  • a human primary cell can be an immune cell.
  • An immune cell can be a T cell, B cell, NK cell, and/or a macrophage.
  • Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells.
  • Suitable cells can comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells. Suitable cells can be progenitor cells. Suitable cells can be derived from the subject to be treated (e.g., a subject with a disease, a subject in need of treatment, or a subject that is immunocompromised). Suitable cells can be derived from a human donor. [00158] A method of attaining suitable cells, such as human primary cells, can comprise selecting cells.
  • a cell can comprise a marker that can be selected for the cell.
  • a marker can comprise GFP, a resistance gene (for example, a gene conferring antibiotic resistance), a cell surface marker, an endogenous tag.
  • Cells can be selected using any endogenous marker.
  • Suitable cells can be selected using any technology. Such technology can comprise flow cytometry and/or magnetic columns.
  • the selected cells can then be infused into a subject.
  • the selected cells can also be expanded to large numbers.
  • the selected cells can be expanded prior to infusion.
  • Delivery vehicles for in vivo and ex vivo use can include pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. 7.
  • compositions comprising a nuclease, nucleic acid editing system, or a fusion protein provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient.
  • a pharmaceutical composition comprising a vector provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient.
  • compositions provided herein e.g., nucleic acid editing systems, nucleases, and fusion proteins provided herein
  • compositions provided herein are combined with pharmaceutically acceptable salts, excipients, and/or carriers to form a pharmaceutical composition.
  • compositions may be chosen based on the route of administration, the location of the target issue, and the time course of delivery of the drug.
  • a pharmaceutically acceptable carrier or excipient may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration.
  • the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol).
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • compositions provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • a dosage unit form is a physically discrete unit of a composition provided herein appropriate for a subject to be treated.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. The animal model is also used to achieve a desirable concentration range and route of administration.
  • compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD 50 (the dose is lethal to 50% of the population).
  • ED50 the dose is therapeutically effective in 50% of the population
  • LD 50 the dose is lethal to 50% of the population.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments.
  • the data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human use.
  • compositions and pharmaceutical compositions for administering to a subject in need thereof are in a form that allows for compositions provided herein to be administered to a subject.
  • the pharmaceutical composition is formulated for intratumoral delivery.
  • administration of a pharmaceutical composition provided herein is local administration or systemic administration.
  • a pharmaceutical composition provided herein is formulated for administration / for use in administration via an intratumoral, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, or intracranial route.
  • the administering is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours. In some embodiments, the administering is daily, weekly, or monthly. In some embodiments, the administering is repeated at least about every 28 days or 56 days.
  • Vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual subject (e.g., lymphocytes, T cells, bone marrow aspirates, or tissue biopsy), followed by reimplantation of the cells into a subject, usually after selection for cells which have incorporated the vector. Prior to or after selection, the cells can be expanded.
  • cells expressing a nuclease, fusion protein, or a nucleic acid editing system provided herein.
  • the cell is contacted in vitro or ex vivo with a nucleic acid encoding for a nuclease, fusion protein, or a nucleic acid editing system provided herein.
  • the cell is contacted in vitro or ex vivo with a vector encoding for an engineered protein provided here nuclease, fusion protein, or a nucleic acid editing system provided herein.
  • the cell is an immune cell.
  • the cell is a lymphocyte, a leukocyte, a T cell, a natural killer cell, a macrophage, a neutrophil, an eosinophil, a basophil, a dendritic cell, a stem cell, a cancer cell, a stem cell, a heart cell, a pancreatic cell, a neuron, an induced pluripotent stem-cell derived cell (iPSC), or an in-vitro differentiated iPSC.
  • the cell is a genetically modified cell.
  • the genetically modified cell is a chimeric antigen receptor (CAR) T cell, an engineered T cell receptor (TCR) T cell, or a tumor-infiltrating lymphocyte (TIL).
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • TIL tumor-infiltrating lymphocyte
  • Cells provided herein can be administered to a subject in need thereof, e.g., a subject with a disease or a condition. Therapeutic applications are discussed further below. 8. Gene Editing Applications & Diagnostics [00169] Provided herein are methods of modifying a target nucleic acid. In some embodiments, the methods comprise, contacting a target nucleic acid molecule with a nuclease, fusion protein, nucleic acid editing system or composition provided herein.
  • the nuclease comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182.
  • the nucleic acid editing system provided herein comprises a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex.
  • the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule.
  • the percentage of target nucleic acid molecule modification is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • the method of modifying the target nucleic acid further comprises mutation of a target nucleic acid sequence.
  • the mutation comprises an insertion or deletion at said one or more nucleic acid sequences.
  • the mutation comprises an indel.
  • the methods further comprise delivering the composition to a cell.
  • the methods further comprise delivering to the cell at least one exogenous nucleic acid molecule for insertion at the cleavage site.
  • the at least one exogenous nucleic acid molecule is delivered with a recombination polypeptide.
  • the recombination polypeptide is an integrase, a flippase, a transponase, or a recombinase.
  • the exogenous nucleic acid molecule comprises at least one sequence having at least 75% homology to the target nucleic acid molecule.
  • the method is in vitro or in vivo. [00171] Provided herein are methods of ex vivo modifying a cell.
  • the methods comprise: contacting a cell with a nuclease, nucleic acid editing system, fusion protein, or a composition provided herein under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell.
  • RNA complex comprises: (i) a nuclease provided herein; and (ii) a guide RNA that binds to a target nucleic acid, wherein upon contacting the cell with the RNP complex, the nuclease cleaves a target nucleic acid molecule, thereby modifying said cell.
  • the cell is an immune cell or a stem cell.
  • the immune cell is a leukocyte, a lymphocyte, a natural killer cell, a dendritic cell, a macrophage, a myeloid cell, a T-cell, a B cell, a stem cell, an induced- pluripotent derived cell, a cancer cell, or an endothelial cell.
  • the stem cell is an embryonic stem cell, an induced-pluripotent stem cell (iPSC), or an adult stem cell.
  • test sample comprises a target nucleic acid capable of binding to the guide nucleic acid.
  • test sample comprises a target nucleic acid capable of binding to the guide nucleic acid.
  • a complex is formed between the guide nucleic acid, a nuclease provided herein, and the target nucleic acid.
  • the nuclease upon formation of the complex the nuclease cleaves the target nucleic acid.
  • the method further comprises detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample.
  • the guide nucleic acid comprises a sequence of any one of: SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152.
  • the nuclease comprises a sequence set forth in any one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182.
  • the method prior to the detecting step, the method further comprises, amplifying the target nucleic acid prior to the detecting step.
  • the amplifying comprises polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), improved multiple displacement amplification (EVIDA), l simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), transcription mediated amplification (TMA), ramification amplification method (RAM), or any combination thereof.
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HDA helicase-dependent
  • the methods further comprise performing an endonuclease mismatch detection assay, an immunoassay, gel electrophoresis, a plasmid interference assay, nucleic acid sequencing, or any combination thereof.
  • the detecting comprises calorimetric detection, potentiometric detection, amperometric detection, optical detection, piezo-electric detection, or any combination thereof.
  • the solid support comprises a reaction chip, a paper, a quartz microfiber, mixed esters of cellulose, a porous aluminum oxide, a patterned surface, a tube, a well, or a matrix. n some embodiments, the solid support comprises a patterned surface suitable for immobilization of molecules in an ordered pattern.
  • a patterned surface refers to an arrangement of different regions in or on an exposed layer of a solid support.
  • the solid support comprises an array of wells or depressions in a surface. The composition and geometry of the solid support can vary with its use.
  • the solid support is a planar structure such as a slide, chip, microchip and/or array. As such, the surface of the substrate can be in the form of a planar layer.
  • the solid support comprises one or more surfaces of a flowcell.
  • a flowcell is a type of chamber comprising a solid surface across which one or more fluid reagents can be flowed.
  • the solid support or its surface is non-planar, such as the inner or outer surface of a tube or vessel.
  • the solid support comprise microspheres or beads.
  • Microspheres, beads, or particles can be made of various material including, but not limited to, plastics, ceramics, glass, and polystyrene.
  • the microspheres are magnetic microspheres or beads.
  • the beads may be porous. The bead sizes range from nanometers, e.g., about 100 nm, to millimeters, e.g., about 1 mm.
  • the test sample comprises a liquid sample.
  • the test sample comprises a biological sample or an environmental sample.
  • the environmental sample comprises a food sample, a paper surface, a fabric, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a saline water sample, or any combination thereof.
  • the environmental sample is suspected of comprising a pathogenic microbial organism. Sequences specific to a pathogen of interest may be identified or selected by comparing the coding sequences from the pathogen of interest to all coding sequences in other organisms by BLAST software.
  • Appropriate samples for use in the methods provided herein include any conventional biological sample obtained from an organism or a part thereof, such as a plant, animal, bacteria, and the like.
  • a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface.
  • a transudate for example, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid
  • a sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ.
  • Exemplary samples include, without limitation, cells, cell lysates, blood smears, cytocentrifuge preparations, cytology smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine-needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).
  • the sample includes circulating tumor cells (which can be identified by cell surface markers).
  • samples are used directly (e.g., fresh or frozen), or can be manipulated prior to use, for example, by fixation (e.g., using formalin) and/or embedding in wax (such as formalin-fixed paraffin-embedded (FFPE) tissue samples).
  • fixation e.g., using formalin
  • FFPE formalin-fixed paraffin-embedded
  • the biological sample comprises a cell extract, cell medium, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a bodily secretion, a transudate, an exudate, a skin swab, or a mucosal membrane swab.
  • the test sample is from a subject that has, is suspected of having, or at risk of developing a disease or a disorder.
  • the nucleic acid editing systems and compositions provided herein have a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. Further provided herein are methods of treating a disease, a disorder, or a condition.
  • diseases and conditions that can be treated using the compositions and methods provided herein include cancer, autoimmune diseases, diabetes, heart disease, infectious diseases, lower respiratory infections, neonatal conditions, neurological diseases, neuromuscular diseases, muscular diseases, gastrointestinal diseases, kidney diseases, liver diseases, vascular diseases, deafness, blindness, and the like.
  • the disease- associated nucleic acid can include RNA transcribed from a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the translated products may be known or unknown, and may be at a normal or abnormal level.
  • the target nucleic acid of a nucleic acid editing system provided herein can be any endogenous or exogenous nucleic acid of a eukaryotic cell.
  • a scaffold comprises: a nuclease provided herein; and a guide nucleic acid provided herein, wherein the nuclease and/or the guide nucleic acid are immobilized to the scaffold.
  • the scaffold comprises a set of nucleases.
  • the set of nucleases comprise at least one nuclease comprising an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77-78, or 182, a functional fragment, or a derivative thereof.
  • the scaffold comprises a set of guide nucleic acids, wherein the set of guide nucleic acids comprise a direct repeat sequence of any one or more of SEQ ID NO: 5 or SEQ ID NO: 6.
  • at least one guide nucleic acid is immobilized to the scaffold.
  • the set of nucleases are immobilized to the scaffold.
  • at least one nuclease and at least one guide nucleic acid are immobilized to the scaffold.
  • at least one nuclease is immobilized to the scaffold and the at least one guide nucleic acid is in complex with the at least one nuclease.
  • the guide nucleic acid comprises a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6; and a variable sequence.
  • the variable sequence is at least 75% complementary to a target sequence.
  • the scaffold further comprises a target sequence in complex with the at least one nuclease and at least one guide nucleic acid.
  • systems comprising a scaffold provided herein.
  • the systems comprise: (a) a scaffold provided herein; (b) a reporter molecule; and (c) a detector.
  • the reporter molecule when a target nucleic acid forms a complex with the scaffold, the reporter molecule produces a detectable signal that is detected by the detector.
  • the system further comprises reagents for nucleic acid amplification.
  • the reporter molecule is selected from the group consisting of: a fluorophore, a dye, a polypeptide, an antibody, a nucleic acid, and any combination thereof.
  • the detectable signal is a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal.
  • kits comprising: a gene editing system provided herein, packaging and materials therefor.
  • a formulation of a composition described herein is prepared in a single container for administration.
  • a formulation of a composition provided herein is prepared two containers for administration, separating the nucleic acids and/or the nucleases provided herein.
  • “container” includes vessel, vial, ampule, tube, cup, box, bottle, flask, jar, dish, well of a single-well or multi-well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and/or transported, and accessed to remove the contents.
  • kits comprising: a first container comprising a nuclease provided herein; and a second container comprising a guide nucleic acid provided herein, packaging and materials therefor. 11.
  • a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 78. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 182.
  • nucleases comprising: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 119, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe.
  • NUC nuclease
  • REC helical recognition
  • nucleases comprising: (a) a nuclease (NUC) lobe comprising a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 20; or SEQ ID NO: 21; and (b) a helical recognition (REC) lobe.
  • NUC nuclease
  • PAM Protospacer Adjacent Motif
  • PI Interacting
  • nucleases comprising: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe.
  • NUC nuclease
  • WED2 wedge 2
  • nucleic acid sequences wherein the nucleic acid sequences encode for a nuclease provided herein.
  • ribonucleoprotein (RNP) complexes wherein the RNP complexes comprises: (i) a nuclease provided herein; and (ii) a guide RNA that binds to a target nucleic acid.
  • vectors wherein the vectors comprise a nucleic acid sequence encoding for a nuclease provided herein.
  • viral vectors wherein the viral vectors comprise a nucleic acid sequence encoding for a nuclease provided herein.
  • nucleic acid editing systems comprising a nuclease provided herein.
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: a nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64 wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease; the guide nucleic acid; and the target nucleic acid form a complex.
  • REC helical recognition
  • nucleic acid editing systems comprise: a nuclease comprising (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC
  • nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
  • nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid.
  • nucleic acid editing systems wherein upon formation of the complex, the nuclease generates a break in the target nucleic acid.
  • nucleic acid editing systems wherein the nuclease generates a single-stranded break in the target nucleic acid or a double-stranded break in the target nucleic acid.
  • nucleic acid editing systems wherein the nuclease generates a double-stranded break with staggered 5’ overhangs in the target nucleic acid.
  • the target nucleic acid is a DNA.
  • nucleic acid editing systems wherein the nuclease further comprises one or more nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • nucleic acid editing systems wherein the one or more NLS is an SV40 Large NLS, a large T antigen NLS, a c-Myc NLS, a nucleoplasmin NLS, an EGL-13 NLS, or a TUS-protein NLS.
  • nucleic acid editing systems wherein the NLS comprises an SV40 Large NLS, and wherein the SV40 Large NLS comprises an amino acid sequence of: MGPKKKRKV (SEQ ID NO: 3). Further provided herein are nucleic acid editing systems, wherein the NLS comprises a nucleoplasmin NLS, and wherein the nucleoplasmin NLS comprises an amino acid sequence of: KRPAATKKAGQAKKKK (SEQ ID NO: 4). Further provided herein are nucleic acid editing systems, wherein the nuclease further comprises: (a) an SV40 Large NLS; and (b) a nucleoplasmin NLS.
  • nucleic acid editing systems wherein the nuclease further comprises: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 63, or SEQ ID NO: 153.
  • nucleic acid editing systems wherein the nuclease further comprises one or more peptide linkers.
  • nucleic acid editing systems wherein the nuclease further comprises a C-terminal tag.
  • nucleic acid editing systems, wherein the C-terminal tag is a c-myc tag.
  • the guide nucleic acid comprises RNA.
  • nucleic acid editing systems wherein the guide nucleic acid comprises RNA and at least one modified nucleoside. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid comprises one or more direct repeat sequences. Further provided herein are nucleic acid editing systems, wherein the one or more direct repeat sequences comprises an sequence of: SEQ ID NO: 5, SEQ ID NO: 6, a functional fragment thereof; or a sequence that is complementary to any one of SEQ ID NO: 5 or SEQ ID NO: 6, or a functional fragment thereof. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid further comprises a sequence that is at least partially complementary to the target nucleic acid sequence.
  • nucleic acid editing systems wherein the guide nucleic acid comprises two direct repeat sequences and a variable sequence, wherein the variable sequence is at least 75% complementary to a target nucleic acid sequence. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid further comprises a promoter sequence. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77.
  • nucleic acid editing systems wherein the nuclease comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77. the nuclease comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78.
  • nucleic acid editing systems wherein the nuclease comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing system is delivered inside a cell or a cellular organelle via electroporation, nucleofection, conjugation, lipofection, calcium phosphate precipitation, or a delivery vehicle.
  • compositions wherein the compositions comprise a delivery vehicle; and a nuclease provided herein or a polynucleotide sequence encoding for a nuclease provided herein. Further provided herein are compositions, wherein the compositions comprise: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof. Further provided herein are compositions, wherein the vector is a viral vector.
  • compositions wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an oncolytic viral vector. Further provided herein are compositions, wherein the compositions further comprise a guide nucleic acid. Further provided herein are compositions, wherein the guide nucleic acid comprises a sequence of any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152.
  • compositions wherein the guide nucleic acid further comprises a sequence that is at least partially complementary to a target nucleic acid sequence.
  • compositions wherein the guide nucleic acid comprises SEQ ID NO: 5 or SEQ ID NO: 6; and a sequence that is at least partially complementary to a target nucleic acid sequence.
  • viral vectors wherein the viral vectors comprise: a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof.
  • viral vectors wherein the viral vectors comprise a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid.
  • viral vectors wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NOS: 5-6.
  • viral vectors comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NOS: 5-9, 36-39, 53-60.
  • compositions wherein the compositions comprise: a viral vector provided herein; and a delivery vehicle.
  • viral vectors and compositions wherein the delivery vehicle is an emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, or a polymer.
  • the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an oncolytic viral vector.
  • lentiviral vectors wherein the lentiviral vectors comprise: a nuclease provided herein, a functional fragment, or a derivative thereof.
  • kits wherein the kits comprise a nuclease provided herein.
  • the kits comprise: a first container comprising a nuclease provided herein; and a second container comprising a guide nucleic acid provided herein, packaging and materials therefor.
  • the kits comprise: a gene editing system provided herein, packaging and materials therefor.
  • a target nucleic acid molecule comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182; and (b) a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex, wherein the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule.
  • the percentage of target nucleic acid molecule modification is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • the methods further comprise mutation of a target nucleic acid sequence.
  • the mutation comprises an insertion or deletion at said one or more nucleic acid sequences.
  • the methods further comprise delivering the composition to a cell.
  • kits for ex vivo modifying a cell comprise: contacting a cell with a composition provided herein under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell.
  • the methods comprise: contacting a cell with a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises: (i) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182; and (ii) a guide RNA that binds to a target nucleic acid, wherein upon contacting the cell with the RNP complex, the nuclease cleaves a target nucleic acid molecule, thereby modifying said cell.
  • the cell is an immune cell or a stem cell.
  • the immune cell is a leukocyte, a lymphocyte, a natural killer cell, a dendritic cell, a macrophage, a myeloid cell, a T-cell, a stem cell, an induced-pluripotent derived cell, a cancer cell, or an endothelial cell.
  • the stem cell is an embryonic stem cell, an induced-pluripotent stem cell (iPSC), or an adult stem cell.
  • the methods comprise: (a) immobilizing a guide nucleic acid onto a solid support; and (b) contacting the guide nucleic acid with: (i) a test sample; and (ii) a nuclease comprising an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182, wherein when the test sample comprises a target nucleic acid capable of binding to the guide nucleic acid, a complex is formed between the guide nucleic acid, the nuclease, and the target nucleic acid, and wherein upon formation of the complex the nuclease cleaves the target nucleic acid; and (c) detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample.
  • the guide nucleic acid comprises a sequence of any one of: SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152. wherein prior to step (c), the method further comprises, amplifying the target nucleic acid.
  • the amplifying comprises polymerase chain reaction (PCR), nucleic acid sequence- based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), improved multiple displacement amplification (EVIDA), l simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), transcription mediated amplification (TMA), ramification amplification method (RAM), or any combination thereof.
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence- based amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HDA helicase-
  • the methods further comprise performing an endonuclease mismatch detection assay, an immunoassay, gel electrophoresis, a plasmid interference assay, nucleic acid sequencing, or any combination thereof.
  • the detecting comprises calorimetric detection, potentiometric detection, amperometric detection, optical detection, piezo-electric detection, or any combination thereof.
  • the solid support comprises a reaction chip, a paper, a quartz microfiber, mixed esters of cellulose, a porous aluminum oxide, a patterned surface, a tube, a well, or a matrix.
  • the test sample comprises a liquid sample.
  • test sample comprises a biological sample or an environmental sample.
  • biological sample comprises a cell extract, cell medium, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a bodily secretion, a transudate, an exudate, a skin swab, or a mucosal membrane swab.
  • NUC lobe further comprises: a wedge 1 (WED1) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 16; or SEQ ID NO: 17; and/or a WED 3 domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 18; or SEQ ID NO: 19.
  • WED1 wedge 1
  • SEQ ID NO: 16 amino acid sequence of: 16
  • SEQ ID NO: 17 or SEQ ID NO: 17
  • WED 3 domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 18; or SEQ ID NO: 19.
  • nuclease wherein the NUC lobe further comprises: a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 20; or SEQ ID NO: 21.
  • PAM Protospacer Adjacent Motif
  • PI Interacting
  • nuclease wherein the NUC lobe further comprises: a RuvCI domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 22; or SEQ ID NO: 23.
  • nuclease wherein the NUC lobe further comprises: a RuvCIII domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 28; or SEQ ID NO: 29. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 11, 2, 10, 11, 77, or 78.
  • nuclease comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence comprising one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78.
  • scaffolds comprising a nuclease or a gene editing system provided herein, wherein the nuclease or the gene editing system is immobilized to the scaffold.
  • scaffolds wherein the set of nucleases comprise at least one nuclease comprising an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof.
  • the scaffolds comprise a set of guide nucleic acids, wherein the set of guide nucleic acids comprise a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • at least one guide nucleic acid is immobilized to the scaffold.
  • the set of nucleases are immobilized to the scaffold.
  • scaffolds wherein at least one nuclease and at least one guide nucleic acid are immobilized to the scaffold. Further provided herein are scaffolds, wherein at least one nuclease is immobilized to the scaffold and the at least one guide nucleic acid is in complex with the at least one nuclease. Further provided herein are scaffolds, wherein the set of guide nucleic acids are each immobilized to the scaffold; and the at least one nuclease is in complex with at least one guide sequence. Further provided herein are scaffolds, wherein at least one nuclease comprises an amino acid sequence with at least 90% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78.
  • scaffolds wherein at least one nuclease comprises an amino acid sequence with at least 95% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are scaffolds, wherein at least one nuclease comprises an amino acid sequence of one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are scaffolds, wherein the guide nucleic acid comprises a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6; and a variable sequence. Further provided herein are scaffolds, wherein the variable sequence is at least 75% complementary to a target sequence.
  • scaffolds wherein the scaffold further comprises a target sequence in complex with the at least one nuclease and at least one guide nucleic acid.
  • systems and devices comprising: (a) a scaffold provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when a target nucleic acid forms a complex with the scaffold, the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector.
  • systems and devices comprising: (a) a nuclease provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when the nuclease cleaves a target nucleic acid the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector.
  • the system further comprises reagents for nucleic acid amplification.
  • the reporter molecule is selected from the group consisting of: a fluorophore, a dye, a polypeptide, an antibody, a nucleic acid, and any combination thereof.
  • the detectable signal is a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal.
  • the fusion proteins comprise a nuclease provided herein.
  • fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonucle
  • fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving
  • fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransfera
  • fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a first nuclease consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a prote
  • isolated proteins wherein the isolated proteins comprise: an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. Further provided herein are isolated proteins, wherein the isolated proteins comprise any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. [00204] Provided herein are isolated nucleic acids, wherein the isolated nucleic acids comprise: a sequence that encodes a protein that comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof.
  • isolated nucleic acids comprising: a sequence that encodes a protein that comprises an amino acid sequence comprising any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof.
  • engineered nucleic acids wherein the engineered nucleic acids comprise: a sequence that is at least 85% identical to SEQ ID NO: 90 or SEQ ID NO: 183.
  • engineered nucleic acids wherein the engineered nucleic acids comprise SEQ ID NO: 90 or SEQ ID NO: 183.
  • Nuclease #1 comprising a sequence of SEQ ID NO: 1
  • Nuclease #2 comprising a sequence of SEQ ID NO: 2 according to guided silencing effects and homolog-specific gRNA cassettes are described herein.
  • 164 proteins were identified to have homology to two known gene editing nuclease proteins. A phylogenetic tree was generated and the percent sequence identity was compared for all sequences ranging from 19% to 50% sequence identity to the known gene editing nuclease protein.
  • Nuclease #1 and Nuclease #2 were generated by standard cloning techniques along with eight other test nuclease sequences.
  • Nuclease #1 and Nuclease #2 were each modified to include an SV40 NLS and nucleoplasmin NLS sequence yielding SEQ ID NO: 10 and SEQ ID NO: 11.
  • Co-factor RNAs were designed using CRISPick software yielding SEQ ID NOS: 5 and 6 for direct repeat sequences.
  • Full length targeting sequences for CD274 and B2M were designed and incorporated into expression cassettes with an hU6 promoter (SEQ ID NO: 31) summarized in Table 6.
  • T7 endonuclease (T7EN1) assays were performed.200 ng of gDNA was used to amplify the genomic locus of CD274 covering the guide targeting sequence.
  • 510 bp PCR amplicons were purified and used for T7 endonuclease assays at 10 ng/ ⁇ L.
  • Samples from the T7EN1 assay were separated by agarose gel electrophoresis to detect the DNA cleavage products.
  • the expected cleavage products were 400 base pairs (bps) and 100 bps using CD274 Guide #1 with control nuclease-CNTRL Nuc.
  • Nuclease #1 and Nuclease #2 produced the expected bands 400 base pair cleavage product (FIG.3).
  • Genomic analysis of B2M and CD274 targeting was performed (FIG. 4A-4B).
  • Nuclease #1 and Nuclease #2 both produced indels/mismatches as using the same CD274 guide sequence (SEQ ID NO: 35).
  • Nuclease #1 produced 1.8% indels targeting CD274 (FIG.4A) and 0.4% indels targeting B2M (FIG.4B).
  • EXAMPLE 2 GUIDE RNA OPTIMIZATION FOR NUCLEASE #1 AND NUCLEASE #2.
  • Optimization of the guide nucleic acid sequence was performed using CRISPick software to improve CD274 targeting by Nuclease #1 (SEQ ID NO: 10) and Nuclease #2 (SEQ ID NO: 11). Different combinations of direct repeat sequences and variable sequences were assayed with their corresponding nucleases.
  • Nuclease #1 and Nuclease #2 were transferred into a suitable lentiviral vector including blasticidin selection (BlastR). Suitable mutations and protein engineering of Nuclease #1 and Nuclease #2 are performed to enhance stability and cleavage efficiency of target DNA. Up to 50 different Nuclease #1 and Nuclease #2 mutants are designed and tested in RKO cells using the protocol outlined in Example 3. Lentiviral vectors comprising the Nuclease #1 or Nuclease #2 mutants are used to establish a pool of 50 to 100 nucleases for a high-throughput screening platform.
  • FIG. 7A-7B Flow cytometry and T7 endonuclease assays were performed (FIG. 7A-7B, FIG. 8, and FIG. 9).
  • Flow cytometry of RKO cells transfected with a mutant nuclease or the WT control nuclease targeting CD274 is shown in FIG. 8A.
  • Double mutant #3 and the triple mutant exhibited a 2-fold reduction in the percentage of CD274 negative RKO cells (approx 40% CD274 negative) relative to the control nuclease with the same guide sequence (FIG. 7A).
  • a T7 endonuclease assay confirmed that the mutant nucleases effectively disrupted the CD274 gene (FIG.7B).
  • Configuration sequences A-C increased CD247 targeting of the control nuclease by over 30% (FIG.10B-10C) and 1.36-fold higher relative to the initial sequence, Configuration 1.
  • Configuration 1 When the triple mutant nuclease (E155R, S532R, K538R) was delivered using configuration A in combination with a CD247-targeting guide sequence, targeting of CD247 was greater than 70% (FIG. 11A). This was also the case for targeting of B2M (FIG. 11B) and CD46 genomic loci (FIG.11C).
  • RKO cells were transfected with one of the guide sequences targeting CD274 and the Triple Mutant Nuclease or the WT control nuclease. Flow cytometry was performed as previously described.
  • the Triple Mutant Nuclease (SEQ ID NO: 90) efficiently targeted TTNN PAMs and NTTN PAMs, indicating that the Triple Mutant Nuclease had greater flexibility for generating double strand breaks in the CD274 gene over the WT control nuclease (SEQ ID NO: 86).
  • the baseline targeting activity of the CD274 guide using a canonical TTTC PAM was used as a reference to rank alternative, non-canonical guides-PAM combinations.
  • Counting of total guide RNAs enabled identification of the most active PAM - guide RNA combinations targeting essential genes as those combinations dropped out resulting in low guide counts (FIG. 14B).
  • the dropout effects improved along the fitness screen as log fold-change (LFC) values were increased on day 21 with PAM sites “CTTC”, “TTTC” and “TTCC” identified as ideal candidates (FIG. 14B).
  • LFC log fold-change
  • three additional PAM sites were identified which can be categorized as “NTTN” (ATTC, ATTG, GTTC).
  • sequences (SEQ ID NOS: 32-35) are formatted to signify the promoter region, the direct repeat (DR) sequence, and variable sequence. Underlined signifies the DR and bold text signifies the variable sequence. Unformatted text is the promoter region of the expression cassette. Docket No.199827-761601/PCT [00241] The following sequences (SEQ ID NOS: 38-39) are formatted to signify the direct repeat (DR) sequence and variable sequence. Bold text signifies the variable sequence. [00242] The following sequences (SEQ ID NO: 53-60) are formatted to signify the direct repeat (DR) sequence and variable sequence. Bold text signifies the variable sequence. SEQ ID NO: 54: Nuclease #1 direct repeat with CD274-targeting guide RNA (bold: Variable Sequence)
  • SEQ ID NOS: 61-62 are formatted to signify the mutation that generates nickase activity. Underlined and bold text signifies the amino acid substitution as compared with reference sequences, SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • SEQ ID NO: 182 The following sequence (SEQ ID NO: 182) is formatted to signify the the amino acid substitutions made in the sequence relative to a reference sequence (e.g., SEQ ID NO: 1). Italicized and bold text signifies the 2A-self-cleaving peptide sequence (SEQ ID NO: 168). Capital letters indicate the endonuclease sequence.
  • SEQ ID NO: 184 The following sequence (SEQ ID NO: 184) is formatted to signify the the nucleic acid substitutions made in the sequence relative to a reference sequence.
  • SEQ ID NO: 184 DNA sequence encoding the guide RNA library scaffold with a GFP - P2A - PuroR - WPRE - hU6 - Optimized direct repeat (DR) (20 bp, bold italics) - Example guide (23 bp) [00247]
  • the following sequence (SEQ ID NO: 185) is formatted to signify the the amino acid substitutions made in the sequence relative to a reference sequence.
  • SEQ ID NO: 185 GFP - P2A (italics)– PuroR (bold and italics) amino acid sequence of the guide RNA library vector.

Abstract

Nucleic acid editing systems, methods, and uses thereof are provided. Various nuclease amino acid sequences and guide RNA sequences are described that promote cleavage of a target nucleic acid sequence. Methods of treating a disease and methods of detecting an alternation in a gene are described. Scaffolds and fusion proteins comprising nuclease mutants and fragments thereof are also provided.

Description

NUCLEIC ACID EDITING SYSTEMS, METHODS, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application No.63/318,906, filed March 11, 2022, and U.S. Provisional Patent Application No. 63/479,274, filed January 10, 2023, the entire contents of which are incorporated herein by reference. SEQUENCE LISTING [0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on March 9, 2023, is named 199827-761601_PCT_SL.xml and is 213,770 bytes in size. BACKGROUND [0003] CRISPR/Cas nucleic acid editing systems have emerged as an approach for the targeted treatment of mammalian diseases and as a tool for diagnostics, synthetic biology, and cell biology. However, current CRISPR/Cas nucleic acid editing systems have low integration rates of exogenous genes for gene-editing applications and some mammals develop Cas-directed immunity that reduces the efficacy of gene editing in mammalian cells. Alternative nucleic acid editing systems are needed for precise genome targeting, selective disruption of individual genetic elements, and reduced innate and adaptive immune responses to programmable nucleases. Such nucleic acid editing systems will advance gene editing capabilities in biotechnology, medical applications, and synthetic biology. BRIEF SUMMARY [0004] The compositions, methods, and kits provided herein are based, in part, on the discovery of new nuclease proteins and nucleic acid editing systems for use in selectively modifying gene expression and gene editing applications. [0005] Provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof. Provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 65. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 67. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 69. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 73. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 75. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 78. In some embodiments, a nuclease provided herein is encoded by a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90. [0006] Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe. Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 15, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe. Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe. [0007] Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe comprising a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 20; and (b) a helical recognition (REC) lobe. Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe comprising a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 21; and (b) a helical recognition (REC) lobe. [0008] Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease is capable of cleaving the target nucleic acid via the NUC lobe. [0009] Further provided herein are nucleic acid sequences, wherein the nucleic acid sequences encode for a nuclease provided herein. Further provided herein are ribonucleoprotein (RNP) complexes, wherein the RNP complexes comprises: (i) a nuclease provided herein; and (ii) a guide RNA that binds to a target nucleic acid. Further provided herein are vectors, wherein the vectors comprise a nucleic acid sequence encoding for a nuclease provided herein. Further provided herein are viral vectors, wherein the viral vectors comprise a nucleic acid sequence encoding for a nuclease provided herein. Further provided herein are nucleic acid editing systems comprising a nuclease provided herein. [0010] Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77; a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. [0011] Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: a nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64; wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease; the guide nucleic acid; and the target nucleic acid form a complex. [0012] Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: a nuclease comprising (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64; wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease; the guide nucleic acid; and the target nucleic acid form a complex. [0013] Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. [0014] Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. [0015] Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 78, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. [0016] Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. [0017] Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. [0018] Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. [0019] Further provided herein are nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of: SEQ ID NO: 5 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of: SEQ ID NO: 6 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid. [0020] Further provided herein are compositions, wherein the compositions comprise a delivery vehicle; and a nuclease provided herein or a polynucleotide sequence encoding for a nuclease provided herein. Further provided herein are compositions, wherein the compositions comprise: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof. In some embodiments, the nucleic acid comprises a sequence that is at least 85% identical to any one of SEQ ID NOS: 85-90, or 183. [0021] Further provided herein are viral vectors, wherein the viral vectors comprise: a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NO: 5 or SEQ ID NO: 6. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152. [0022] Further provided herein are kits, wherein the kits comprise a nuclease provided herein. Further provided herein are kits, wherein the kits comprise: a first container comprising a nuclease provided herein; and a second container comprising a guide nucleic acid provided herein, packaging and materials therefor. Further provided herein are kits, wherein the kits comprise: a gene editing system provided herein, packaging and materials therefor. [0023] Further provided herein are methods of modifying a target nucleic acid molecule, wherein the methods comprise: contacting a target nucleic acid molecule with a composition comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182; and (b) a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex, wherein the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule. [0024] Further provided herein are methods of ex vivo modifying a cell, wherein the methods comprise: contacting a cell with a composition provided herein under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell. [0025] Further provided herein are methods of ex vivo modifying a cell, wherein the methods comprise: contacting a cell with a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises: (i) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182; and (ii) a guide RNA that binds to a target nucleic acid, wherein upon contacting the cell with the RNP complex, the nuclease cleaves a target nucleic acid molecule, thereby modifying said cell. [0026] Further provided herein are methods of detecting one or more target nucleic acid molecules in a test sample, wherein the methods comprise: (a) immobilizing a guide nucleic acid onto a solid support; and (b) contacting the guide nucleic acid with: (i) a test sample; and (ii) a nuclease comprising an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182; wherein when the test sample comprises a target nucleic acid capable of binding to the guide nucleic acid, a complex is formed between the guide nucleic acid, the nuclease, and the target nucleic acid, and wherein upon formation of the complex the nuclease cleaves the target nucleic acid; and (c) detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample. [0027] Further provided herein are uses for a nuclease provided herein. Further provided herein are uses for a nuclease provided herein, wherein the nuclease comprises: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe. [0028] Further provided herein are scaffolds comprising a nuclease or a gene editing system provided herein, wherein the nuclease or the gene editing system is immobilized to the scaffold. Further provided herein are scaffolds, wherein the scaffolds comprise: (a) a set of nucleases, wherein at least one nuclease comprises an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182, a functional fragment, or a derivative thereof; and (b) a set of guide nucleic acids, wherein the set of nucleases and/or the set of guide nucleic acids are immobilized to the scaffold. [0029] Further provided herein are systems and devices comprising: (a) a scaffold provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when a target nucleic acid forms a complex with the scaffold, the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector. Further provided herein are systems and devices comprising: (a) a nuclease provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when the nuclease cleaves a target nucleic acid the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector. [0030] Further provided herein are fusion proteins, wherein the fusion proteins comprise a nuclease provided herein, a fragment, or a derivative thereof. Further provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, a zinc finger, or any combination thereof, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [0031] Further provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, a zinc finger, or any combination thereof, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [0032] Further provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182, a functional fragment, or a derivative thereof; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, a zinc finger, or any combination thereof, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [0033] Further provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a first nuclease consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, a zinc finger, or any combination thereof, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [0034] Provided herein are isolated proteins, wherein the isolated proteins comprise: an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. Further provided herein are isolated proteins, wherein the isolated proteins comprise any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. [0035] Provided herein are isolated nucleic acids, wherein the isolated nucleic acids comprise: a sequence that encodes a protein that comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof. Further provided herein are isolated nucleic acids, wherein the isolated nucleic acids comprise: a sequence that encodes a protein that comprises an amino acid sequence comprising any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof. [0036] Provided herein are engineered nucleic acids, wherein the engineered nucleic acids comprise: a sequence that is at least 85% identical to SEQ ID NO: 90 or SEQ ID NO: 183. Further provided herein are engineered nucleic acids, wherein the engineered nucleic acids comprise SEQ ID NO: 90 or SEQ ID NO: 183. A BRIEF DESCRIPTION OF THE DRAWINGS [0037] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0038] FIGURES 1A-1B shows a schematic representation of a gene editing system. FIG. 1A shows a nuclease provided herein, including a NUC lobe, a REC lobe, and associated domains. FIG. 1B shows a gRNA provided herein including a direct repeat (DR) sequence that is a scaffold for the nuclease and a variable sequence that is at least partially complementary to a target nucleic acid. [0039] FIGURES 2A-2C show nucleic acid editing system targeting of CD274 (also called PD-L1). FIG. 2A shows a schematic representation of the protocol used to determine nuclease targeting in RKO cells. FIG. 2B shows flow cytometry analysis of CD274 expression in cells transfected with DNA encoding Nuclease #1 without a guide RNA (CNTRL Nuc) and Nuclease #1 (SEQ ID NO: 10) with CD274-targeting gRNA, SEQ ID NO: 53. FIG. 2C shows a graph demonstrating the percentage (%) of CD274 negative cells for control nuclease-targeted cells and additional test nucleases as indicated with a CD274-targeting gRNA. [0040] FIGURE 3 shows a T7 endonuclease assay demonstrating indel formation for control cells (not transfected with a nuclease), a CNTRL Nuc, test nucleases, and Nuclease #1 and Nuclease #2 as indicated. Cleavage products are indicated with arrows. [0041] FIGURE 4A-4B shows graphs demonstrating of the percentage and identity of indels produced when cells were treated with an CNTRL Nuc-gRNA; Nuclease #2-gRNA; and Nuclease #1-gRNA. FIG. 4A shows indel/mismatch % for nucleases and a CD274-targeting gRNA (SEQ ID NO: 53). Next generation sequencing (NGS) results for Nuclease #1 are shown. FIG. 4B shows indel/mismatch % for nucleases and a B2M-targeting gRNA (SEQ ID NO: 38). Next generation sequencing (NGS) results for Nuclease #1 are also shown. FIG. 4A discloses SEQ ID NOS: 173-179, and FIG.4B discloses SEQ ID NOS: 180-181 and 154, respectively, in order of appearance. [0042] FIGURE 5 shows flow cytometry analysis of RKO cells contacted with Nuclease #1 and guide nucleic acids SEQ ID NOS: 53-56. [0043] FIGURE 6 shows flow cytometry analysis of RKO cells contacted with Nuclease #2 and guide nucleic acids SEQ ID NOS: 57-60. [0044] FIGURES 7A-7B show a graph of flow cytometry and gel electrophoresis after T7 endonuclease assay for RKO cells transfected with a guide sequence targeting CD274 and a control or mutant nuclease. FIG.7A is a graph showing the percentage of CD247 negative RKO cells for each of the following conditions: (1) CNTRL Nuclease (SEQ ID NO: 1), no guide; (2) CNTRL Nuclease + Guide; (3) REC Mutant # 1 + Guide (E155R, SEQ ID NO: 65); (4) WED2 Mutant #1 + Guide (S532R, SEQ ID NO: 67); (5) WED2 Mutant #2 + Guide (K538R, SEQ ID NO: 69); (6) Double Mutant #1 + Guide (E155R, S532R, SEQ ID NO: 71); (7) Double Mutant #2 + Guide (E155R, K538R, SEQ ID NO: 73); (8) Double Mutant #3 + Guide (S532R, K538R, SEQ ID NO: 75); and (9) Triple Mutant + Guide (E155R, S532R, K538R, SEQ ID NO: 77). FIG. 7B is a T7 endonuclease assay demonstrating indel formation for control cells (not transfected with a nuclease); a CNTRL Nuclease (SEQ ID NO: 1), no guide; CNTRL Nuclease + Guide; REC Mutant # 1 + Guide (E155R, SEQ ID NO: 65); WED2 Mutant #1 + Guide (S532R, SEQ ID NO: 67); WED2 Mutant #2 + Guide (K538R, SEQ ID NO: 69); Double Mutant #1 + Guide (E155R, S532R, SEQ ID NO: 71); Double Mutant #2 + Guide (E155R, K538R, SEQ ID NO: 73); Double Mutant #3 + Guide (S532R, K538R, SEQ ID NO: 75); and Triple Mutant + Guide (E155R, S532R, K538R, SEQ ID NO: 77). [0045] FIGURE 8 shows flow cytometry results for RKO cells transfected with a guide sequence targeting CD46 and a control or mutant nuclease. The percentage of CD46 negative RKO cells is shown for each of the following conditions: (1) CNTRL Nuclease (SEQ ID NO: 1), no guide; (2) CNTRL Nuclease (SEQ ID NO: 1) + Guide; (3) REC Mutant # 1 (SEQ ID NO: 65) + Guide (E155R); (4) WED2 Mutant #1 (SEQ ID NO: 67) + Guide (S532R); (5) WED2 Mutant #2 (SEQ ID NO: 69) + Guide (K538R); (6) Double Mutant #1 (SEQ ID NO: 71) + Guide (E155R, S532R); (7) Double Mutant # 2 (SEQ ID NO: 73) + Guide (E155R, K538R); (8) Double Mutant #3 (SEQ ID NO: 75) + Guide (S532R, K538R); and (9) Triple Mutant + Guide (E155R, S532R, K538R, SEQ ID NO: 77). [0046] FIGURE 9 shows flow cytometry results for RKO cells transfected with a guide sequence targeting B2M and a control or mutant nuclease. The percentage of B2M negative RKO cells is shown for each of the following conditions: 1) CNTRL Nuclease (SEQ ID NO: 1), no guide; (2) CNTRL Nuclease (SEQ ID NO: 1) + Guide; (3) REC Mutant # 1 (SEQ ID NO: 65) + Guide (E155R); (4) WED2 Mutant #1 (SEQ ID NO: 67) + Guide (S532R); (5) WED2 Mutant #2 (SEQ ID NO: 69) + Guide (K538R); (6) Double Mutant #1 (SEQ ID NO: 71) + Guide (E155R, S532R); (7) Double Mutant # 2 (SEQ ID NO: 73) + Guide (E155R, K538R); (8) Double Mutant #3 (SEQ ID NO: 75) + Guide (S532R, K538R); and (9) Triple Mutant + Guide (E155R, S532R, K538R, SEQ ID NO: 77). [0047] FIGURES 10A-10C show a schematic and assay results for various nuclear localization sequence configurations for the control nuclease. FIG. 10A shows a schematic of Configuration 1 (SEQ ID NO: 85), Configuration A (SEQ ID NO: 86), Configuration B (SEQ ID NO: 87), and Configuration C (SEQ ID NO: 88). FIG.10B shows the percentage of CD247 negative RKO cells when cells were contacted with a Configuration 1 construct, Configuration A construct, a Configuration B construct, or a Configuration C construct. FIG. 10C shows flow cytometry graphs for each sequence configuration. [0048] FIGURES 11A-11C show graphs of the flow cytometry assay results for various nuclear localization sequence configurations comparing the WT nuclease using the NLS configuration 1 (SEQ ID NO: 85) with the optimized Triple Mutant nuclease using the NLS configuration A (SEQ ID NO: 90). FIG. 11A shows a graph of the percentage of CD274 negative RKO cells. FIG. 11B shows the percentage of B2M negative RKO cells. FIG. 11C shows the percentage of CD46 negative RKO cells after co-transfection with the respective sgRNAs. [0049] FIGURE 12 shows the NGS results of RKO cells co-transfected with the WT (SEQ ID NO: 85) or Triple Mutant nucleases (SEQ ID NOS: 89 and 90) and sgRNAs targeting either B2M, CD274 or CD46. Genomic DNA was extracted from transfected RKO cells and genomic loci of B2M, CD274 and CD46 genes were PCR amplified. PCR amplicons of 200 bp, 210 bp or 233 bp were sequenced via MiSeq PE150 paired-end reads. The NGS data was used to quantify the percentage of reads with indels for each genomic target locus indicating that the triple mutant nuclease with configuration A (SEQ ID NO: 90) scored up to 65-85% indel formation (genomic editing). RKO cells not transfected with guides or nucleases were used as negative controls (RKO CNTRL and WT CNTRL Nuclease). [0050] FIGURES 13A-13B show the effect of various PAM sequences on nuclease targeting of CD274. FIG. 13A show a graph of the % of CD247- negative RKO cells treated with the control nuclease and guide sequences targeting CD247. FIG. 13B show a graph of the % of CD247- negative RKO cells treated with the Triple Mutant nuclease and guide sequences targeting CD247. [0051] FIGURES 14A-14B show graphs demonstrating Triple Mutant nuclease fitness effects relative to the starting point on day 0. FIG.14A shows a graph of fitness effects assessed on day 7 relative to day 0 (guide RNA library representation) FIG.14B shows a graph of fitness effects on day 21 (final time point) post guide RNA transduction. The dropout effects of the fitness screen are shown as log fold-change (LFC) values relative to day 0 (X-axis). PAM sites (e.g., “CTTC”, “TTTC” and “TTCC”) are shown on the Y-axis. [0052] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art. DETAILED DESCRIPTION [0053] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. [0054] Provided herein are compositions, kits, methods, and uses thereof for editing a target gene. Briefly, further described herein are (1) nucleases; (2) guide nucleic acids; (3) target nucleic acids; (4) fusion proteins; (5) nuclease activity and efficiency; (6) delivery systems and vectors; (7) pharmaceutical compositions, dosing, and administration; (8) gene editing applications and diagnostics; (9) scaffolds and systems; and (10) kits. Definitions [0055] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. [0056] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. All references disclosed herein, including patent references and non-patent references, are hereby incorporated by reference in their entirety as if each was incorporated individually. However, where a patent, patent application, or publication containing express definitions is incorporated by reference, those express definitions should be understood to apply to the incorporated patent, patent application, or publication in which they are found, and not necessarily to the text of this application, in particular the claims of this application, in which instance, the definitions provided herein are meant to supersede. [0057] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” [0058] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. [0059] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. [0060] As used herein, "optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. [0061] The term “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value. For example, the amount “about 10” includes amounts from 9 to 11. The term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. [0062] The term “cell” or “engineered cell” and their grammatical equivalents as used herein refers to a cell of human or non-human origin. [0063] The term “cleavage” and its grammatical equivalents as used herein refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond, and introducing 5’ base pair (bp) staggered cuts. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In some embodiments, fusion polypeptides are used for targeted single-stranded RNA or single-stranded DNA cleavage. In some embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage. In some cases, cleavage refers to processing of an array of guide nucleic acids, such that individual guide nucleic acids are created. Such processing can involve recognition of a direct repeat region of a guide sequence and cleavage upstream of the direct repeat region. [0064] The term “delivery vehicle” refers to a formulation when in combination with a composition provided herein (e.g., a nuclease) delivers the composition to a cell. The delivery vehicle can be a solid, semi-solid, a liquid, or a gas. In some embodiments, a delivery vehicle provided herein is man-made or a non-naturally occurring vehicle. In some embodiments, a delivery vehicle is naturally occurring. For example, a delivery vehicle can comprise a vesicle, an exosome, a liposome, or a cell. In some embodiments, a delivery vehicle provided herein is isolated from a biological fluid. The type of delivery vehicle used can be determined by a skilled practitioner and will depend on the use of the compositions provided herein. [0065] The term “effective amount” or “therapeutically effective amount” refers to an amount that is sufficient to achieve or at least partially achieve the desired effect. [0066] The term “engineered” and its grammatical equivalents as used herein refers to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome. The term “engineered” refers to alterations, additions, and/or deletion of genes. An engineered cell can also refer to a cell with an added, deleted and/or altered gene. [0067] The term “function” and its grammatical equivalents as used herein refers to the capability of operating, having, or serving an intended purpose. Functional can comprise any percent from baseline to 100% of normal function. For example, functional can comprise or comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, and/or 100% of normal function. In some embodiments, the term functional can mean over or over about 100% of normal function, for example, 125, 150, 175, 200, 250, 300% and/or above normal function. [0068] The term “functional fragment” and its grammatical equivalents as used herein refers to a protein, polypeptide or nucleic acid whose sequence is not identical to the full- length protein, polypeptide or nucleic acid, yet retains the same or similar function as the full- length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid, or cleavage of a nucleic acid) include, but are not limited to: genomic Cleavage Detection assays, RT-PCR, protein expression assays, fluorescence in situ hybridization (FISH), reporter splice-correction assays, cellular in vitro assays and animal models. [0069] The term “gene editing” and its grammatical equivalents as used herein refers to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g., a naturally-existing nuclease or an artificially engineered nuclease) or other methods known to those of ordinary skill in the art. [0070] The term “homolog” and its grammatical equivalents as used herein refers to a protein of the same organism which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related. [0071] The terms “modifying”, “altering”, or “disrupting” and their grammatical equivalents as used herein refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof. Modifying a gene can, for example, partially or completely suppress expression of the gene. Modifying a gene can also cause activation of a different gene, for example, a downstream gene. [0072] The term “modulation” and its grammatical equivalents as used herein refers to a change in the activity or level of a given parameter (e.g., gene expression). For example, modulation of gene expression can include, but is not limited to, gene activation and gene repression. [0073] The term “mutation” and its grammatical equivalents as used herein can include the substitution, deletion, and insertion of one or more nucleotides in a polynucleotide. For example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence can be substituted, deleted, and/or inserted. A mutation can affect the coding sequence of a gene or its regulatory sequence. A mutation can also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA and/or the translated polypeptide structure and function [0074] The term “nuclease” and its grammatical equivalents as used herein refers to a naturally occurring or engineered construct or protein that can be guided by a single guide nucleic acid molecule to specifically recognize a target nucleic acid comprising a sequence that binds the guide nucleic acid molecule. A nuclease has nuclease activity or the ability to cleave a phosphodiester bond in a target nucleic acid. [0075] The term “ortholog” and its grammatical equivalents as used herein refers to a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. [0076] The term “percent complementary” and its grammatical equivalents as used herein refers to the percentage of complementary base-pairs in a heteroduplex molecule. Complementary base-pairs are known in the art, and their complementarity is achieved by distinct interaction between nucleobases, e.g., adenine (A) pairs with thymine (T) or uracil (U); and guanine (G) pairs with cytosine (C). The term “partially complementary” refers to a nucleic acid sequence (e.g., a variable sequence of a guide nucleic acid) having a substantially complementary sequence to another nucleic acid sequence (e.g., a target nucleic acid sequence) but that differs from the other nucleic acid by at least two or more nucleotides. As such, a partially complementary nucleic acid specifically excludes a population containing sequences that are exactly complementary, that is, a complementary sequence that has 100% complementarity. Therefore, each member of such a partially complementary nucleic acid population differs from other members of the population by two or more nucleotides, including both strands. 1. Nucleases [0077] Provided herein are nucleases, nucleic acid editing systems, and methods for use in modifying or altering a target nucleic acid sequence. In some embodiments, the nucleases provided herein can be used in various therapeutic, diagnostic, and biotechnological applications as described herein. The nucleic acid editing systems provided herein have high specificity for a target nucleic acid, high efficacy, and high safety. The nucleases provided herein are guided by a single guide nucleic acid (e.g., a guide RNA) that binds downstream of a protospacer adjacent motif (PAM). Binding is mediated by full or partial complementarity of the variable region of the guide RNA. A PAM is a short nucleic acid sequence (usually 2-6 base pairs in length) that precedes the region targeted for cleavage by the nuclease provided herein. Upon binding to a target nucleic acid via the guide sequence, the nucleases provided herein specifically cut the target nucleic acid (e.g., DNA) at the distal end of the PAM, by introducing staggered cuts. The nucleases and nucleic acid editing systems provided herein have several advantages over other gene editing systems including, but not limited to the following: (1) the nucleases provided herein do not require a trans-activating crRNA (tracrRNA); (2) the guide nucleic acid is shorter than guide RNAs typically used for other nucleases (e.g., Cas9); (3) the nucleases provided herein are capable of processing a multi-cistronic guide RNA that enables multiplexed gene editing; (4) the nucleases provided herein induce staggered ends when cutting the target nucleic acid that enables higher integration rates of exogenous nucleic acids, improving gene editing efficiency; and (5) the nucleases provided herein can be used as a gene therapy and can optionally be used to avoid Cas9-directed immunity in vivo. [0078] Provided herein are nucleases that form a complex with a guide nucleic acid and a target nucleic acid provided herein. Further provided herein is a nuclease with DNA cleavage activity. [0079] The nucleases provided herein comprise a helical recognition (REC) lobe that recognizes the target nucleic acid and a nuclease (NUC) lobe, which cuts the target nucleic acid. A schematic representation of the structure of a nuclease provided herein is shown in FIG. 1. In some embodiments, the NUC lobe comprises a wedge 1 (WED 1) domain; a WED 2 domain; a Protospacer Adjacent Motif (PAM) Interacting (PI) domain; a WED 3 domain; a RuvC1 domain; a bridge helix; a RuvC2 domain; a nuclease (Nuc); and a RuvC3 domain. In some embodiments, the nuclease comprises a REC lobe comprising a REC1 domain and a REC2 domain. The RuvC domain of the nucleases provided herein comprise three split RuvC motifs: RuvC I, RuvC II, and RuvC III. The nucleases provided herein lack a HNH endonuclease domain. In addition, a nuclease (NUC) lobe provided herein comprises a helical region between RuvC I and RuvC II, and a zinc finger-like domain between RuvC II and RuvC III. [0080] In some embodiments, a nuclease provided herein comprises an amino acid sequence that is least 85% identical to an amino acid sequence listed in Table 1. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to an amino acid sequence listed in Table 1. In some embodiments, a nuclease provided herein comprises an amino acid sequence selected from Table 1. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NOS: 64-78, or SEQ ID NO: 182. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NOS: 64-78 or SEQ ID NO: 182. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is 100% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NOS: 64-78 or SEQ ID NO: 182. In some embodiments, a nuclease provided herein comprises at least one of SEQ ID NOS: 12 to 29, 40, 41, 66, 68, 70, 72, 74, 76 or any combination thereof. Percent (%) sequence identity for a given sequence relative to a reference sequence is defined as the percentage of identical residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity. Percent identity can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software such as BLAST, Align, ClustalW2. Table 1. Nuclease Amino Acid Sequences.
Figure imgf000022_0001
[0081] In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 12 and SEQ ID NO: 14. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 12 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 14 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 13 and SEQ ID NO: 15. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 13 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 15 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 27. [0082] In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 66 and SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 66 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 64 and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 66, SEQ ID NO: 14, and SEQ ID NO: 26. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 68 and SEQ ID NO: 15. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 68 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 15 and SEQ ID NO: 27. In some embodiments, a nuclease provided herein comprises: SEQ ID NO: 68, SEQ ID NO: 15, and SEQ ID NO: 27. [0083] Additional combinations of nuclease amino acid sequences can include but are not limited to the sequences provided in Table 2. The amino acid sequences in Table 2 can be combined to generate a functional nuclease provided herein (e.g., a nuclease that cleaves a target nucleic acid). In some embodiments, a nuclease provided herein comprises at least two amino acid sequences selected from Table 2, in any combination. Table 2: Nuclease Amino Acid Sequence Combinations.
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
[0084] Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is derived from one or more bacteria. In some embodiments, the nuclease is a chimeric nuclease, wherein the chimeric nuclease comprises an amino acid sequence that is derived from two or more different bacteria. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived or isolated from a bacterium of the genus Prevotella. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Prevotella ruminicola bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a P. bryantii bacterium, a P. brevis bacterium, or a P. albensis bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Prevotella ruminicola, wherein the Prevotella ruminicola is of the strain ATCC 19189 / JCM 8958 / 23. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived or isolated from a bacterium of the genus Rumminococcus. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Rumminococcus bovis bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Rumminococcus bovis, wherein the Rumminococcus bovis is of the strain ATCC 10801. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus sp. AF37-3AC bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus bromii bacterium. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus sp. AM36-18. In some embodiments, a nuclease provided herein comprises an amino acid sequence that is derived from a Ruminococcus sp. AM28-29LB. [0085] In some embodiments, a nuclease provided herein further comprises a nuclear localization sequence (NLS). In some embodiments, the nuclease comprises more than one nuclear localization sequence (NLS). An NLS targets a protein to the nucleus of a cell, localizing a nuclease provided herein in close proximity to a target nucleic acid within the nucleus of a cell. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to an NLS sequence listed in Table 3, a functional fragment, or a derivative thereof. In some embodiments, the NLS comprises an amino acid sequence that is 100% identical to an NLS sequence listed in Table 3, a functional fragment, or a derivative thereof. Table 3. Nuclear Localization Sequences.
Figure imgf000027_0001
Figure imgf000028_0001
[0086] In some embodiments, a nuclease provided herein comprises an SV40 NLS. In some embodiments, a nuclease provided herein comprises a nucleoplasmin nuclear localization sequence. In some embodiments, a nuclease provided herein comprises an SV40 NLS and a nucleoplasmin nuclear localization sequence. In some embodiments, a nuclease provided herein comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 63, or SEQ ID NO: 153. [0087] In some embodiments, a nuclease provided herein comprises a linker. A linker is a molecular entity that can directly or indirectly connect at two parts of a composition, e.g., from the first protein construct to the second protein construct and the second protein construct to a third protein construct, and so on. Linkers can be configured according to a specific need, e.g., stability or length between two amino acid sequences. In some embodiments, linkers can be configured to allow multimerization of at least two nucleases provided herein (e.g., to from a di-, tri-, tetra-, penta-, or higher multimeric complex) while retaining biological activity (e.g., cleavage of a target nucleic acid or set of target nucleic acids). In some embodiments, linkers can be configured to facilitate expression and purification of the nuclease provided herein. [0088] In some embodiments, a linker can be configured to have any length in a form of a peptide, peptidomimetic, a protein, a nucleic acid (e.g., DNA or RNA), or any combinations thereof. In some embodiments, the linker can vary from about 2 to about 4 amino acids long, from about 2 to about 6 amino acids long, from about 2 to about 10 amino acids long, or from about 2 to about 100 amino acids long. Longer or shorter linker sequences can be also used for the nucleases and fusion proteins provided herein. [0089] In some embodiments, the linker can be configured to have a sequence comprising at least one of the amino acids selected from the group consisting of glycine (Gly), serine (Ser), asparagine (Asn), threonine (Thr), methionine (Met) or alanine (Ala). In some embodiments, uncharged polar amino acids (e.g., Gln, Cys, or Tyr)or nonpolar amino acids (e.g., Val, Leu, Pro, Phe, and Trp), can also be included in a linker sequence. In some embodiments, polar amino acids can be included. [0090] The linkers can be of any shape. In some embodiments, a linker can be linear. In some embodiments, a linker can be folded. In some embodiments, a linker can be branched. In some embodiments, a linker further comprises a detectable label. [0091] In some embodiments, a linker can be a chemical linker of any length. In some embodiments, chemical linkers can comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NH, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms, such as substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substituted or unsubstituted C5-C12 heterocyclyl, substituted or unsubstituted C3-C12 cycloalkyl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, NH, or C(O). In some embodiments, the chemical linker can be a polymer chain (branched or linear). [0092] In some embodiments, the linker is a GSGSGS linker (SEQ ID NO: 52). In some embodiments, there can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers or more on a polypeptide construct. For example, there can be from 1 to 10 GSGSGS linkers. A linker can comprise non-charged or charged amino acids. A linker can comprise alpha-helical domains. A linker can comprise a chemical cross linker. In some embodiments, a linker can be of different lengths to adjust the function of fused domains and their physical proximity. In some embodiments, a linker can comprise peptides with ligand-inducible conformational changes. [0093] In some embodiments, a nuclease provided herein further comprises a tag. In some embodiments, the tag is a C-terminal tag. In some embodiments, the tag is an N-terminal tag. The tag provided herein can be used to detect the nuclease in a sample or immobilize the nuclease to a solid support (e.g, a column). In some embodiments, the tag is a c-myc tag. In some embodiments, the tag is a recombinant protein. Non-limiting examples of protein tags include: His tags (e.g., HHHHHH (SEQ ID NO: 155)), chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag (WSHPQFEK) (SEQ ID NO: 156), and glutathione-S-transferase (GST). In some embodiments, the tag is a solubilization tag (e.g., thioredoxin (TRX), polyNANP, MBP, and GST). In some embodiments, the tag is a FLAG-tag (DYKDDDDK (SEQ ID NO: 157)). In some embodiments, the tag is an epitope tag. Non-limiting examples of epitope tags include: an ALFA-tag (SRLEEELRRRLTE (SEQ ID NO: 158)), a V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 159)), a Myc-tag (EQKLISEEDL (SEQ ID NO: 30)), an HA-tag (YPYDVPDYA (SEQ ID NO: 160)), a Spot-tag (PDRVRAVSHWSS (SEQ ID NO: 161)), a T7-tag (MASMTGGQQMG (SEQ ID NO: 162)) and a NE-tag (TKENPRSNQEESYDDNES (SEQ ID NO: 163)). In some embodiments, the tag is a HiBiT- tag. In some embodiments, the tag is an Fc tag, a Nus-tag, a Biotin Carboxyl Carrier Protein (BCCP) tag, an SPB tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 164)), a Ty tag (EVHTNQDPLD (SEQ ID NO: 165)), an Xpress tag (DLYDDDDK (SEQ ID NO: 166)). In some embodiments, the tag is a horseradish peroxidase (HRP), a chloramphenicol acetyltransferase (CAT) beta-galactosidase, a beta-glucuronidase, or a luciferase. [0094] In some embodiments, the tag is a fluorescent tag or a fluorophore. Non-limiting examples of fluorescent tags include: red fluorescent protein, cjBlue, aeBlue, amilGFP, amilCP, Green fluorescent protein (GFP), mCherry, mOrange, blue fluorescent protein (BFP), yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), Venus, Cerulean, HcRed, DsRed, among many others. Any tag that facilitates the detection or immobilization of a nuclease provided herein or a guide nucleic acid provided herein can be used. Methods of detecting a protein include without limitation, Western blotting techniques, immunochemistry, immunosorbent assays (e.g., ELISA), enzyme activity assays (e.g. reporter assays), mass spectrometry, colorimetric assays, luciferase assays, and proteomics. [0095] In some embodiments, a nuclease provided herein comprises at least one amino acid substitution or deletion. In some embodiments, a nuclease provided herein comprises at least one amino acid substitution or deletion within the nuclease domain of the nuclease amino acid sequence. In some embodiments, the amino acid substitution or deletion is between position 1067 and 1262 of the nuclease amino acid sequence. In some embodiments, the amino acid substitution or deletion occurs at position 1165 or position 1186. In some embodiments, the amino acid substitution is an R1165A substitution. In some embodiments, the amino acid substitution is R1186A substitution. In some embodiments, a nuclease is modified to become a catalytically dead nuclease or a partially dead nuclease. In some embodiments, a nuclease comprises SEQ ID NO: 61 or SEQ ID NO: 62, a functional fragment, or a derivative thereof. [0096] In some embodiments, the amino acid substitution is within the WED2 domain of a nuclease provided herein. In some embodiments, the amino acid substitution is within the REC1 domain of a nuclease provided herein. In some embodiments, a nuclease provided herein comprises one or more amino acid substitution. In some embodiments, a nuclease provided herein comprises two or more amino acid substitutions. In some embodiments, a nuclease provided herein comprises three or more amino acid substitutions. In some embodiments, a nuclease provided herein comprises an amino acid substitution at glutamate 155 (Glu155, E155), serine 532 (Ser 532, S532), lysine 538 (Lys 538, K538), or any combination thereof as compared to the sequence of SEQ ID NO: 1. In some embodiments, a nuclease provided herein comprises an amino acid substitution at E155, S532, and/or K538, wherein the amino acid substitution at any one of positions E155, S532, and/or K538 comprises a substitution of an amino acid residue to a different amino acid residue comprising a hydrophobic amino acid residue, a hydrophilic amino acid residue, a charged amino acid residue that is a basic amino acid residue, or an acidic amino acid residue, or an aliphatic amino acid residue. In some embodiments, the different amino acid residue comprises a charged amino acid residue that is a basic amino acid residue. In some embodiments, the different amino acid residue is an arginine (Arg, R) or a lysine (Lys, K), a glutamate (Glu, E), an aspartate (Asp, D), or a valine (Val, V). In some embodiments, a nuclease provided herein comprises an amino acid substitution listed in Table 8. In some embodiments, a nuclease provided herein comprises an amino acid sequence of any one of SEQ ID NOS: 64-78. In some embodiments, a nuclease provided herein is encoded by any one of the nucleic acid sequences of SEQ ID NOS: 85-90, or 183. In some embodiments, a nuclease provided herein is encoded by a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90, or 183. Methods of modifying a nucleic acid sequence encoding a nuclease include, e.g., direct mutagenesis or random mutagenesis. 2. Guide Nucleic Acids [0097] Provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise a guide nucleic acid. In some embodiments, a nuclease provided herein binds to a guide nucleic acid provided herein. In some embodiments, a guide nucleic acid is a guide DNA (gDNA) or a guide RNA (gRNA). In some embodiments, the guide nucleic acid comprises RNA. In some embodiments, the guide nucleic acid comprises RNA and/or DNA. [0098] In some embodiments, the guide nucleic acid comprises RNA and a non- canonical nucleobase or non-canonical nucleoside. Canonical nucleobases include, for example, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). Nucleoside analogues are non-canonical nucleosides which contain a nucleobase analogue and a sugar. Once phosphorylated, nucleosides transition to nucleotides capable of incorporation into growing RNA strands. While non-canonical nucleosides are capable of incorporating into a newly synthesized nucleic acid strand, they are generally not capable of being “read” by an RNA polymerase for amplification and act as chain terminators that stop an RNA polymerase. Modified nucleobases which can be incorporated into modified nucleosides and nucleotides and be present in the guide nucleic acid include: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6- methyladenosine), s2U (2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6- hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2 ′ -O-ribosyladenosine (phosphate)); I (inosine); m1I (1- methylinosine); m′ Im (1,2′ -O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O- methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2 ′ -O- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine); Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); hoSU (5-hydroxyuridine); moSU (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5- methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5- methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5- carbamoylmethyl-2 ′ -O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U (5- carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2′ -O- methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5- hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2 ′ 7G (N2,7- dimethylguanosine); m2 ′ 2 ′ 7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O- dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2 ′ -O-methylcytidine); m1Gm (1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1- C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5- (hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1- C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8- azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8- substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino- 6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2′-O-methyl-U. Any one or any combination of these modified nucleobases may be included in the guide sequence. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers. If desired, the guide nucleic acid can contain phosphoramidate, phosphorothioate, and/or methylphosphonate linkages. [0099] Guide nucleic acids provided herein that comprise at least one modified nucleotide can be prepared using any suitable method. Several suitable methods are known in the art for producing nucleic acid molecules that contain modified nucleotides. For example, a guide nucleic acid that contains modified nucleotides can be prepared by transcribing (e.g., in vitro transcription) a DNA that encodes for a guide RNA using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and the like, or mutants of these polymerases which allow efficient incorporation of modified nucleotides into RNA molecules. The transcription reaction can contain nucleotides and modified nucleotides, and other components that support the activity of the selected polymerase, such as a suitable buffer, and suitable salts. The incorporation of nucleotide analogs into a guide nucleic acid may be engineered, for example, to alter the stability of such RNA molecules or to increase resistance against RNases. [00100] In some embodiments, the guide nucleic acid is from about 1 base pair to about 30 base pairs in length. In some embodiments, the guide nucleic acid is at least partially complementary to a target nucleic acid sequence. In some embodiments, the guide nucleic acid binds a target nucleic acid. In some embodiments, the guide nucleic acid is at least about 75% complementary to a target polynucleotide sequence. In some embodiments, the guide nucleic acid is at least about 80% , 85%, 90%, 95%, 99% complementary to a target nucleic acid sequence. In some embodiments, a guide nucleic acid is fully or 100% complementary to a target nucleic acid sequence. [00101] Guide nucleic acids provided herein can comprise a direct repeat sequence. A direct repeat sequence is generally within 20 to 30 base pairs (bps) in length. In some embodiments, the direct repeat sequence is at least about 16 bps in length or more, 17 bps in length or more, 18 bps in length or more, 19 bps in length or more, 20 bps in length or more, 21 bps in length or more,, 22 bps in length or more, 23 bps in length or more, 24 bps in length or more, 25 bps in length or more, 26 bps in length or more, 27 bps in length or more, 28 bps in length or more, 29 bps in length or more, 30 bps in length or more. In some embodiments, guide nucleic acids provided herein comprise one or more, two or more, three or more, or four or more direct repeat sequences. The length and sequence composition of the guide nucleic acid provided herein may influence the binding of the nuclease provided herein to the guide nucleic acid and the target nucleic acid. [00102] In some embodiments, the direct repeat sequence comprises a short stem-loop structure. In some embodiments, the direct repeat sequence comprises a sequence of SEQ ID NO: 5 or SEQ ID NO: 6, a functional fragment, or a derivative thereof. In some embodiments, the guide nucleic acid comprise SEQ ID NO: 5 or SEQ ID NO: 6; and a sequence that is at least partially complementary to a target nucleic acid sequence. Exemplary guide nucleic acids and their corresponding targets and nucleases are provided in Table 4 below. Additional guide sequences are also provided in Table 13 based on the target sequence protospacer adjacent motif (PAM). Table 4: Exemplary Guide Nucleic Acids.
Figure imgf000034_0001
Figure imgf000035_0001
[00103] The direct repeat sequences operably linked to the variable sequences in Table 4, Table 9, and Table 13, are set forth in SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. [00104] In some embodiments, the guide nucleic acid comprises a sequence that is at least 85% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. In some embodiments, the guide nucleic acid comprises a sequence that is at least 90% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. In some embodiments, the guide nucleic acid comprises a sequence that is at least 95% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. In some embodiments, the guide nucleic acid comprises a sequence that is at least 99% identical to a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. In some embodiments, the guide nucleic acid comprises a sequence set forth in any one of SEQ ID NOS: 38, 39, 53-60, 79-84, 113-152. [00105] In some embodiments, the guide nucleic acid comprises a variable sequence targeting a nucleic acid encoding for CD274, wherein the variable sequence comprises any one of SEQ ID NOS: 7-9, 36, 79, 113-152. In some embodiments, the guide nucleic acid comprises any one of SEQ ID NOS: 53-60, or 113-152. In some embodiments, the guide nucleic acid comprises a variable sequence targeting a nucleic acid encoding for B2M, wherein the variable sequence comprises SEQ ID NO: 37 or SEQ ID NO: 81. In some embodiments, the guide nucleic acid comprises any one of SEQ ID NOS: 38-39, SEQ ID NO: 84. In some embodiments, the guide nucleic acid comprises a variable sequence targeting a nucleic acid encoding for cluster of differentiation 46 (CD46), wherein the variable sequence comprises SEQ ID NO: 80. In some embodiments, the guide nucleic acid comprises SEQ ID NO: 83. In some embodiments, the In some embodiments, the guide nucleic acid comprises a sequence of any one of: SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152. [00106] Further provided herein are nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid. [00107] In some embodiments, the guide nucleic acid comprises a variable sequence. In some embodiments, the variable sequence is at least partially complementary to a target nucleic acid sequence. In some embodiments, guide nucleic acids provided herein comprise a direct repeat sequence and a variable sequence. In some embodiments, a nucleic acid editing system provided herein comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5. In some embodiments, a nucleic acid editing system provided herein comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5. In some embodiments, a nucleic acid editing system provided herein comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6. In some embodiments, a nucleic acid editing system provided herein comprises a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6. [00108] In some embodiments, guide nucleic acids provided herein are delivered to a cell via an expression cassette. An expression cassette is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a nucleic acid sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a nucleic acid to be transcribed, operably linked to a promoter. [00109] In some embodiments, guide nucleic acids provided herein further comprise a promoter sequence. The promotor sequence can be any promoter that permits expression of the guide RNA in a cell. For example, a promoter capable of transcribing the guide RNA in a cell includes a heat shock /heat inducible promoter operably linked to a nucleotide sequence encoding the guide RNA. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. Non-limiting examples of promotors include: a ubiquitin promoter (e.g., U6); a Pol-II promoter; a Pol-III promoter; a Banana Streak Virus Promoter (BSV promoter); a Phospholipid Transfer Protein Promoter (PLTP promoter); H1, EF-1a, and a T7 promoter. [00110] In some embodiments, guide nucleic acids provided herein comprise a promoter sequence, a direct repeat sequence, and a variable sequence. In some embodiments, the guide nucleic acid optionally, comprises a polynucleotide linker. In some embodiments, the guide nucleic acid comprises two direct repeat sequences and a variable sequence, wherein the variable sequence is at least 75% complementary to a target nucleic acid sequence. [00111] Guide nucleic acids provided herein may comprise non-naturally occurring sequences or engineered sequences. A guide nucleic acid sequence can comprise synthetic RNA or DNA molecules. Furthermore, guide nucleic acids can be generated by transducing or transfecting cells with a plasmid DNA that encodes for an expression cassette. For example, a U6 promoter can be used to drive the expression of the guide RNA. In some embodiments, the U6 promoter comprises the sequence of SEQ ID NO: 31. 3. Target Nucleic Acids [00112] A guide nucleic acid provided herein can be at least partially complementary to a target polynucleotide sequence of interest. In some embodiments, a target nucleic acid provided herein comprises a gene sequence. In some embodiments, the gene sequence is a mammalian gene sequence. In some embodiments, the gene sequence is a human gene sequence. In some embodiments, a target nucleic acid provided herein comprises a DNA. In some embodiments, a target nucleic acid provided herein comprises a double-stranded DNA. In some embodiments, a target nucleic acid provided herein comprises a single-stranded DNA. In some embodiments, a target nucleic acid provided herein comprises RNA. In some embodiments, a target nucleic acid provided herein comprises single stranded RNA. In some embodiments, a target nucleic acid provided herein comprises double stranded RNA. In some embodiments, a target nucleic acid binds to a variable sequence of a guide nucleic acid. In some embodiments, the target nucleic acid comprises a protospacer-adjacent motif ("PAM"), wherein the PAM is a short T-rich sequence. In some embodiments, cleavage of a target nucleic acid occurs downstream from the PAM sequence. Molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In some embodiments, the PAM is a T-rich PAM. In some embodiments, the PAM has the nucleotide sequence (T)XN, wherein the X is the number of thymines (e.g, 1-10), and N is A, G, C or T. In certain embodiments, X is equal to 2, and thus, the PAM is TTN. In some embodiments, X is 3, and thus, the PAM is TTTN. In some embodiments, a nuclease provided herein recognizes the sequence motif TTTN and directs cleavage of a target nucleic acid sequence 1-24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) bp downstream from that sequence. In some embodiments, the PAM site is a TTTA sequence or a TTTC sequence. In some embodiments, the PAM site is a sequence listed in Table 13. [00113] Without limitation, the target nucleic acid sequence can be from any cell or organism. Determining the appropriate sequence for the guide nucleic acid to bind to a target nucleic acid provided herein may depend on the sequence of the target and structure of the nuclease provided herein. Methods of designing a guide nucleic acid for gene editing are known in the art and include, e.g., using software and databases such as Breaking-Cas, Cas-OFFinder, CRISPR-DT, CHOPCHOP, CCTOP, CRISPick, or CRISPOR. In some embodiments, the target nucleic acid sequence is a mammalian DNA sequence. In some embodiments, the target nucleic acid sequence is a human DNA sequence. In some embodiments, the target nucleic acid sequence is a sequence expressed by a tumor or a cancer cell. In some embodiments, the target nucleic acid comprises a sequence associated with a disease or condition. In some embodiments, the target nucleic acid sequence is a sequence expressed by an immune cell. Non-limiting examples of target nucleic acids include nucleic acid encoding: cluster of differentiation 274 (CD274, also called programmed cell death ligand 1 or PD-L1), beta-2-microglobulin (B2M), CD46, CD81, C-C chemokine receptor type 5 (CCR5), BAF Chromatin Remodeling Complex Subunit 11 (BCL11A), transthyretin (TTR), programmed cell death 1 (PD-1), programmed cell death 2 (PD-2), T cell immunoglobulin and ITIM domain (TIGIT), Lymphocyte Activating 3 (LAG3), and Cytokine-inducible SH2-containing protein (CISH). 4. Fusion proteins [00114] Provided herein are fusion proteins comprising a nuclease provided herein. The nucleases provided herein can be fused to one or more additional protein constructs for a particular application (e.g., therapeutic gene editing or cellular gene editing for biotechnology applications in vitro or ex vivo ). Multimerization of an effector or protein tag by direct fusion with the nuclease provided herein can localize the effector domain to a site matched by the guide nucleic acid provided herein. The fusion proteins provided herein can be used to modulate transcription, expression, translation, RNA splicing, methylation status, DNA unwinding, or any other property that alters nucleic acid expression in a cell. [00115] In some embodiments, nucleases provided herein are fused to at least one additional element. In some embodiments, a fusion protein comprises a first protein construct comprising a nuclease provided herein. In some embodiments, a fusion protein provided herein comprises a second protein construct. In some embodiments, fusion proteins provided herein optionally, comprise a linker between the first protein construct and second protein construct. In some embodiments, fusion proteins provided herein further comprise a third, fourth, and/or fifth protein construct. In some embodiments, a fusion protein provided herein comprises one or more linkers between one or more protein constructs. In some embodiments, a fusion protein provided herein comprises a cleavable or non-cleavable linker between the different protein constructs or domains of the fusion protein. For example, a linker can be a polypeptide linker, such as a linker that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids long. As described herein, two polypeptide constructs that are “fused” need not be directly adjacent to each other. Fused polypeptide sequences can be fused by a linker, or by an additional functional or non-functional polypeptide sequence that is fused to the polypeptide sequences. Generally, the number of additional elements in the nuclease-fusion protein will depend on a number of factors, including, e.g., size or molecule weight of the fusion protein for delivery to a cell, or translocation of the fusion protein desired in a cell type of interest. [00116] In some embodiments, a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof. In some embodiments, a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof. In some embodiments, a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof. In some embodiments, a fusion proteins provided herein comprise a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 99% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof. In some embodiments, a fusion protein provided herein comprises a first protein construct comprising: a nuclease comprising an amino acid sequence that is 100% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, 182, a functional fragment, or a derivative thereof. [00117] In some embodiments, a fusion protein provided herein comprises one or more amino acid sequence selected from Table 1. In some embodiments, a fusion protein provided herein comprises: a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14; or SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid. In some embodiments, the first protein construct comprises a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid. In some embodiments, the first protein construct (e.g., a first nuclease) is capable of cleaving the target nucleic acid via the NUC lobe. [00118] In some embodiments, a fusion protein provided herein comprises a second protein construct. In some embodiments, a fusion protein provided herein comprises a protein construct selected from Table 5. Table 5 provides a list of proteins that can be fused to a nuclease provided herein, a functional fragment, or a derivative thereof. Table 5 also provides the function of various proteins and exemplary NCBI/UniProt accession numbers, which can be selected using ordinary skill in the art, for instance, based on amino acid sequence, crystal structure data, computational structure predictions, and functional activity. Table 5. Protein constructs.
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
[00119] In some embodiments, a fusion protein provided herein comprises a protein construct comprising an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 5, a functional fragment, or a derivative thereof. In some embodiments, a fusion protein provided herein comprises a protein construct comprising a protein from Table 5, a homolog, or an ortholog thereof. Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling, e.g., BLAST, NCBI, or UniProt. In some embodiments, the additional protein construct is of eukaryotic origin, such as of human, rat or lamprey origin. In some embodiments, the additional protein construct is of prokaryotic origin, such as a bacterium or archaeon. In some embodiments, the additional protein construct comprises a viral protein. [00120] In some embodiments, a fusion protein provided herein comprises one or more additional protein constructs, wherein the one or more additional protein constructs comprises: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, a zinc finger, and any combination thereof. [00121] In some embodiments, a fusion protein provided herein comprise a Cas protein. In some embodiments, the Cas is a catalytically dead Cas or a partially dead Cas (e.g., a nickase). In some embodiments, the catalytically dead Cas or the partially dead Cas is selected from the group consisting of catalytically dead derivatives of: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, xCas9, CasX, CasY, and CasRX [00122] In some embodiments, the fusion protein provided herein comprises a nickase, wherein the nickase comprises a sequence that is at least 85% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nickase comprises a sequence that is at least 90% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nickase comprises a sequence that is at least 95% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nickase comprises a sequence comprising SEQ ID NO: 61. In some embodiments, the nickase comprises a sequence comprising SEQ ID NO: 62. [00123] In some embodiments, the fusion protein provided herein comprises a nuclease deficient protein construct, wherein the nuclease deficient protein construct comprises a sequence that is at least 85% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nuclease deficient protein construct comprises a sequence that is at least 90% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nuclease deficient protein construct comprises a sequence that is at least 95% identical to SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the nuclease deficient protein construct comprises a sequence comprising SEQ ID NO: 61. In some embodiments, the nuclease deficient protein construct comprises a sequence comprising SEQ ID NO: 62. [00124] In some embodiments, a fusion protein provided herein comprises a protein construct that modulates transcription. In some embodiments, a fusion protein provided herein comprises a transcriptional repressor protein construct. In some embodiments, a fusion protein provided herein comprises a zinc finger protein construct. In some embodiments, the zinc finger protein construct comprises a Krüppel-associated box (KRAB) protein or a functional fragment thereof. In some embodiments, the fusion protein comprises a KRAB domain that binds to a transcriptional corepressor protein. In some embodiments, a fusion protein provided herein comprises a SUMO protein construct. [00125] In some embodiments, a fusion protein provided herein comprises a transcriptional activator protein construct. A transcriptional activator protein construct recruits transcription factors from a host cell to the target nucleic acid for regulation of target nucleic acid expression. Exemplary transcriptional activators include VP64, VP16, VP160, VP48, VP96, p65, Rta, VPR, hsf1, and p300. In some embodiments, a fusion protein provided herein comprises one or more VP16 protein construct. In some embodiments, a fusion protein provided herein comprises one or more VP64 protein construct. In some embodiments, a fusion protein provided herein comprises one or more VPR protein construct. In some embodiments, a fusion protein provided herein comprises one or more SunTag protein construct. In some embodiments, a fusion protein provided herein comprises VP64, p65, and HSF1 (SunTag-p65-heat-shock factor 1 or SPH). In some embodiments, a fusion protein provided herein comprises a CREB-binding protein (CPB). CBP can be used to recruit transcriptional machinery and function as a histone acetyltransferase (HAT) that alters chromatin structure. [00126] In some embodiments, a nuclease or a fusion protein provided herein further comprises an aptamer. In some embodiments, the aptamers are bind to one or more MS2 proteins. [00127] In some embodiments, a fusion protein provided herein comprises a self-cleaving protein sequence. Non-limiting examples of self-cleaving protein sequences include: E2A, P2A and T2A. In some embodiments, the fusion protein comprises an antibiotic resistance protein construct or an antibiotic resistance selectable marker. Non-limiting examples of antibiotic resistance proteins and selectable markers include: aminoglycoside acetyltransferase, rifampin ADP-ribosyltransferase, dihydrofolate reductase, multidrug and toxic compound extrusion transporters, antibiotic resistance ATP-binding cassette family F (ARE ABC-F) proteins (e.g., MsrE, Erm, Vga, Lsa, Sal, OptrA), β-lactamase, blasticidin-S deaminase, penicillin-binding proteins (PBPs), and puromycin-N-acetyltransferase. [00128] In some embodiments, a fusion protein provided herein comprises a base editor. In some embodiments, the base editor is selected from the group consisting of: an adenosine nucleobase editor, a cytidine deaminase, an adenine base editor. In some embodiments, the cytidine deaminase is activation-induced deaminase (AID), an APOBEC deaminase, APOBEC3G, APOBEC1, cytidine deaminase 1 (CDA1), a functional fragment, or a derivative thereof. In some embodiments, the adenosine base editor is ecTadA, saTadA, a functional fragment, or a derivative thereof. In some embodiments, fusion proteins provided herein further comprise a uracil-DNA glycosylase, a functional fragment, or a derivative thereof. [00129] Further provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a first nuclease consisting of SEQ ID NO: 1, 2, 10, 11, 77-78; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [00130] Fusion proteins provided herein can be synthesized using known technologies, for instance, recombination DNA technology where the coding sequences of various portions of the fusion proteins can be linked together at the nucleic acid level. Subsequently a fusion protein can be produced using a host cell. 5. Methods of Determining Gene Editing Efficiency [00131] Provided herein are methods of determining gene editing efficiency and selectivity of a nuclease or fusion protein provided herein for a target nucleic acid sequence. In some embodiments, the target nucleic acid is cleaved by a nuclease provided herein upon binding of the guide sequence to the target nucleic acid sequence. In some embodiments, the target nucleic acid is a DNA. In some embodiments, the nuclease provided herein cleaves the DNA resulting in a double-stranded break (DSB). In some embodiments, the nuclease provided herein cleaves the target DNA via staggered DNA double-stranded break. In some embodiments, the staggered DNA double-stranded break generates a 5' overhang. The 5' overhang can facilitate gene insertion via non-homologous end joining (NHEJ) mechanisms. In some embodiments, the 5’ overhang is at least 3 base pairs (bps) or more, at least 4 bps or more, at least 5 bps or more, or at least 6 bps or more. In some embodiments, the 5’ overhang is about 4 bps to 5 bps in length. The ability of a nuclease to recognize a PAM sequence can be determined by an in vitro selection assay. [00132] The activity of a nucleic acid editing system may be assayed using a cell expressing a reporter protein or containing a reporter gene. For example, a reporter protein may be engineered to contain an obstruction, such as a stop codon, a frameshift mutation, a spacer, a linker, or a transcriptional terminator; the nucleic acid editing system may then be used to remove the obstruction and the resultant functional reporter protein may be detected. In some embodiments, the obstruction may be designed such that a specific sequence modification is required to restore functionality of the reporter protein. In other embodiments, the obstruction may be designed such that any insertion or deletion which results in a frame shift of one or two bases may be sufficient to restore functionality of the reporter protein. Examples of reporter proteins include colorimetric enzymes, metabolic enzymes, fluorescent proteins, enzymes and transporters associated with antibiotic resistance, and luminescent enzymes. Examples of such reporter proteins include β-galactosidase, Chloramphenicol acetyltransferase, Green fluorescent protein, Red fluorescent protein, luciferase, and renilla. Different detection methods may be used for different reporter proteins. For example, the reporter protein may affect cell viability, cell growth, fluorescence, luminescence, or expression of a detectable product. In some embodiments, the reporter protein may be detected using a colorimetric assay. In some embodiments, the reporter protein may be a fluorescent protein, and DNA editing may be assayed by measuring the degree of fluorescence in treated cells, or the number of treated cells with at least a threshold level of fluorescence. In some embodiments, transcript levels of a reporter gene may be assessed. In other embodiments, a reporter gene may be assessed by sequencing. In some embodiments, an assay for measuring DNA editing may use a split fluorescence protein system, such as the self-complementing split GFP1-10/11 systems, in which two fragments (G1-10 and G11) of the GFP protein which can associate by themselves to form a functional GFP signal are linked using a frameshifting linker. Insertions or deletions within the frameshifting linker can restore the frame of the G11 fragment allowing the two fragments to form a functional GFP signal. Integration of an exogenous nucleic acid can be measured using any technique, e.g., integration can be measured by flow cytometry, surveyor nuclease assay, tracking of indels by decomposition (TIDE), junction PCR, or any combination thereof. In other embodiments, transgene integration can be measured by PCR. A TIDE analysis can also be performed on engineered cells. Ex vivo cell transfection can also be used for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism). In some embodiments, cells are isolated from the subject organism, transfected with a nucleic acid (e.g., gene or cDNA), and re-infused back into the subject organism (e.g., subject). [00133] The amount of nuclease-containing modified cells that can be necessary to be therapeutically effective in a subject can vary depending on the viability of the cells, and the efficiency with which the cells have been genetically modified (e.g., the efficiency with which a transgene has been integrated into one or more cells). In some embodiments, the product (e.g., multiplication) of the viability of cells post genetic modification and the efficiency of integration of a transgene can correspond to the therapeutic aliquot of cells available for administration to a subject. In some embodiments, an increase in the viability of cells post genetic modification can correspond to a decrease in the amount of cells that are necessary for administration to be therapeutically effective in a subject. In some embodiments, an increase in the efficiency with which a transgene has been integrated into one or more cells can correspond to a decrease in the amount of cells that are necessary for administration to be therapeutically effective in a subject. In some embodiments, determining an amount of cells that are necessary to be therapeutically effective can comprise determining a function corresponding to a change in the viability of cells over time. In some embodiments, determining an amount of cells that are necessary to be therapeutically effective can comprise determining a function corresponding to a change in the efficiency with which a transgene can be integrated into one or more cells with respect to time dependent variables (e.g., cell culture time, electroporation time, cell stimulation time). [00134] Non-homologous end joining (NHEJ) and homology-directed repair (HDR) can be quantified using a variety of methods. For example, a percent of NHEJ, HDR, or a combination of both can be determined by co-delivering the gene editing molecules, for example a guide nuclease acid and a nuclease or fusion protein provided herein, with a donor nucleic acid template that encodes a promoter-less tag or marker (e.g., GFP) into cells. After a duration of time (e.g., 72-96 hours), flow cytometry can be performed to quantify the total cell number (NTotal), tag-positive cell number and tag/GFP-negative cell number. Among the tag-negative cells, next-generation sequencing can be performed to identify cells without mutations and with mutations. HDR efficiency and NHEJ efficiency can be calculated from the assay. [00135] Additional assays for determining gene editing efficiency of a nucleic acid editing system for cleaving a nucleic acid include but are not limited to: RT-PCR, nucleic acid sequencing, T7 endonuclease 1 (T7E1) mismatch detection assays, tracking of indels by decomposition (TIDE) assays, and indel detection by amplicon analysis (IDAA) assays. The indel pattern that is induced at the target site of a programmable nuclease may also be determined by PCR-amplifying the respective region and subsequent next generation sequencing. To obtain a quantitative single-cell view of gene editing efficiency, we targeted cell surface markers (e.g., CD274 or B2M) and quantified the loss of signal that occurs as a consequence of indel formation by flow cytometry. 6. Delivery Systems [00136] Provided herein are polynucleotides encoding a nuclease provided herein. In some embodiments, the polynucleotide encoding a nuclease provided herein comprises a promoter sequence. Useful promoters for nuclease encoding sequences include, e.g., CMV, EF- 1a, MSCV, PGK, and CAG control promoters. [00137] Further provided herein are compositions, wherein the compositions comprise: a nucleic acid encoding for a nuclease provided herein; and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: one or more nucleic acids encoding for a nucleic acid editing system provided herein and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: one or more nucleic acids encoding for a fusion protein provided herein and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for a nuclease provided herein; a second nucleic acid encoding for a guide nucleic acid provided herein; and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for a fusion protein provided herein; a second nucleic acid encoding for a guide nucleic acid provided herein; and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: a first nucleic acid encoding for a first protein construct provided herein; a second nucleic acid encoding for a second protein construct; a third nucleic acid encoding for a guide nucleic acid provided herein; and a delivery vehicle. Further provided herein are compositions, wherein the compositions comprise: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77-78, or 182, a functional fragment, or a derivative thereof. In some embodiments, the composition further comprises an exogenous nucleic acid for delivery to a cell. In some embodiments, the exogenous nucleic acid is incorporated into a target nucleic acid sequence. In some embodiments, the exogenous nucleic acid encodes for a protein construct provided in Table 5. [00138] The nucleases, fusion proteins, guide nucleic acids, and nucleic acid editing systems provided herein can be delivered to a target cell by any suitable means. The nucleases and guide nucleic acids provided herein can be admixed with a delivery vehicle that permits delivery of the system to the target nucleic acid sequence. Non-limiting examples of delivery vehicles include an emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, a nanoparticle, a polymer, microcapsules, recombinant cells, cell culture medium, blood, or serum. Specific types of delivery vehicles that can be used in a composition provided herein are further described below. [00139] In some embodiments, the delivery vehicle is a liposome. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core. Liposomes interact with cells via different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one can operate at the same time. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles can also be used as a delivery vehicle. [00140] In some embodiments, the delivery vehicle is a phospholipid. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the liposomes form. Physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less- ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs. [00141] In some embodiments, the delivery vehicle is a nanoparticle. Nanoparticle carriers that specifically target a tissue provided herein may also be used as a pharmaceutically acceptable carrier. In some embodiments, the nanoparticle is a gold nanoparticle, a platinum nanoparticle, an iron-oxide nanoparticle, a lipid nanoparticle, a selenium nanoparticle, a tumor- targeting glycol chitosan nanoparticle (CNP), a cathepsin B sensitive nanoparticle, a hyaluronic acid nanoparticle, a paramagnetic nanoparticle, or a polymeric nanoparticle. [00142] The nucleic acid editing systems provided herein can be delivered using vectors, for example containing sequences encoding one or more of the nucleases or fusion proteins provided herein. In some embodiments, a nucleic acid editing system as described herein can be delivered absent a viral vector. Transgenes encoding polynucleotides can be similarly delivered. Any vector systems can be used including, but not limited to, plasmid vectors, viral vectors, and oncolytic viral vectors. Furthermore, any of these vectors can comprise one or more transcription factor, nuclease, and/or transgene. [00143] In some embodiments, vectors provided herein are a viral vector. Exemplary viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adeno- associated viral vectors (AAV), adenoviral vectors, herpes simplex viral vectors, alphaviral vectors, flaviviral vectors, rhabdoviral vectors, measles viral vectors, Newcastle disease viral vectors, poxviral vectors, and picornaviral vectors. In some embodiments, polynucleotides encoding an engineered protein provided herein are contained in an AAV viral vector. In some embodiments, vectors provided herein are an oncolytic viral vector. In some embodiments, polynucleotides encoding for a nuclease or a fusion protein provided herein are contained in an lentiviral viral vector. Provided herein are lentiviral vectors, wherein the lentiviral vectors comprise a polynucleotide encoding for a nucleic acid editing system provided herein. [00144] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding for a nuclease provided herein, a nucleic acid editing system, and/or transgenes in cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding protein constructs and fusion proteins provided herein and/or transgenes to cells in vitro. In some embodiments, nucleases, fusion proteins, and gene editing systems provided herein are administered for in vivo or ex vivo uses. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. [00145] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by AMAXA® Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. (see for example U.S. Pat. No. 6,008,336). Lipofection reagents are sold commercially (e.g., TRANSFECTAM® and LIPOFECTIN®). Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. [00146] Vectors including viral and non-viral vectors containing nucleic acids encoding a nucleic editing system provided herein can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA or mRNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. More than one route can be used to administer a particular composition. [00147] In some embodiments, a vector encoding for an exogenous transgene can be shuttled to a cellular nucleus. For example, a vector can contain a nuclear localization sequence (NLS). An NLS can be from Simian Vacuolating Virus 40. A vector can also be shuttled by a protein or protein complex. In some embodiments, a nuclease provided herein can be used as a means to shuttle a minicircle vector. In some embodiments, the exogenous transgene comprises one or more NLS. In some embodiments, a vector can be pre-complexed with a nuclease provided herein prior to electroporation into a cell. A nuclease that can be used for shuttling can be a nuclease-deficient or catalytically dead protein. A nuclease that can be used for shuttling can be a nuclease-competent protein. In some embodiments, a nuclease provided herein can be pre-mixed with a guide nucleic acid and a vector or plasmid encoding an exogenous transgene. [00148] A cell can be transfected with a mutant or chimeric adeno-associated viral vector encoding an exogenous transgene and a nucleic acid editing system comprising a nuclease provided herein. An AAV vector concentration can be from 0.5 nanograms to 50 micrograms. The amount of nucleic acid (e.g., ssDNA, dsDNA, and/or RNA) that can be introduced into the cell by electroporation can be varied to optimize transfection efficiency and/or cell viability. In some embodiments, less than about 100 picograms of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated). In some embodiments, at least about 100 picograms, at least about 200 picograms, at least about 300 picograms, at least about 400 picograms, at least about 500 picograms, at least about 600 picograms, at least about 700 picograms, at least about 800 picograms, at least about 900 picograms, at least about 1 microgram, at least about 1.5 micrograms, at least about 2 micrograms, at least about 2.5 micrograms, at least about 3 micrograms, at least about 3.5 micrograms, at least about 4 micrograms, at least about 4.5 micrograms, at least about 5 micrograms, at least about 5.5 micrograms, at least about 6 micrograms, at least about 6.5 micrograms, at least about 7 micrograms, at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14 micrograms, at least about 15 micrograms, at least about 20 micrograms, at least about 25 micrograms, at least about 30 micrograms, at least about 35 micrograms, at least about 40 micrograms, at least about 45 micrograms, or at least about 50 micrograms, of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated). For example, 1 microgram of dsDNA can be added to each cell sample for electroporation. In some embodiments, the amount of nucleic acid (e.g., dsDNA) required for optimal transfection efficiency and/or cell viability can be specific to the cell type. In some embodiments, the amount of nucleic acid (e.g., dsDNA) used for each sample can directly correspond to the transfection efficiency and/or cell viability. [00149] The transfection efficiency of cells with any of the nucleic acid delivery platforms described herein, for example, nucleofection or electroporation, can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%. [00150] Viral particles, such as AAV, can be used to deliver a viral vector comprising a gene of interest or a transgene into a cell ex vivo or in vivo. In some embodiments, a mutated or chimeric adeno-associated viral vector as disclosed herein can be measured as pfu (plaque forming units). In some embodiments, the pfu of recombinant virus or mutated or chimeric adeno-associated viral vector of the compositions and methods of the disclosure can be about 108 to about 5×1010 pfu. In some embodiments, recombinant viruses of this disclosure are at least about 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, and 5×1010 pfu. In some embodiments, recombinant viruses of this disclosure are at most about 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, and 5×1010 pfu. In some aspects, a mutated or chimeric adeno-associated viral vector of the disclosure can be measured as vector genomes. In some embodiments, recombinant viruses of this disclosure are 1×1010 to 3×1012 vector genomes, or 1×109 to 3×1013 vector genomes, or 1×108 to 3×1014 vector genomes, or at least about 1×101, 1×102, 1×103, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, and 1×1018 vector genomes, or are 1×108 to 3×1014 vector genomes, or are at most about 1×101, 1×102, 1×103, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, and 1×1018 vector genomes. [00151] In some embodiments, a mutated or chimeric adeno-associated viral vector of the disclosure can be measured using multiplicity of infection (MOI). In some embodiments, MOI can refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic can be delivered. In some embodiments, the MOI can be 1×106 GC/mL. In some embodiments, the MOI can be 1×105 GC/mL to 1×107 GC/mL. In some embodiments, the MOI can be 1×104 GC/mL to 1×108 GC/mL. In some embodiments, recombinant viruses of the disclosure are at least about 1×101 GC/mL, 1×102 GC/mL, 1×103 GC/mL, 1×104 GC/mL, 1×105 GC/mL, 1×106 GC/mL, 1×107 GC/mL, 1×108 GC/mL, 1×109 GC/mL, 1×1010 GC/mL, 1×1011 GC/mL, 1×1012 GC/mL, 1×1013 GC/mL, 1×1014 GC/mL, 1×1015 GC/mL, 1×1016 GC/mL, 1×1017 GC/mL, and 1×1018 GC/mL MOI. In some embodiments, a mutated or chimeric adeno-associated viruses of this disclosure are from about 1×108 GC/mL to about 3×1014 GC/mL MOI, or are at most about 1×101 GC/mL, 1×102 GC/mL, 1×103 GC/mL, 1×104 GC/mL, 1×105 GC/mL, 1×106 GC/mL, 1×107 GC/mL, 1×108 GC/mL, 1×109 GC/mL, 1×1010 GC/mL, 1×1011 GC/mL, 1×1012 GC/mL, 1×1013 GC/mL, 1×1014 GC/mL, 1×1015 GC/mL, 1×1016 GC/mL, 1×1017 GC/mL, and 1×1018 GC/mL MOI. [00152] In some aspects, a non-viral vector or nucleic acid can be delivered without the use of a mutated or chimeric adeno-associated viral vector and can be measured according to the quantity of nucleic acid. Generally, any suitable amount of nucleic acid can be used with the compositions and methods of this disclosure. In some embodiments, nucleic acid can be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g. In some embodiments, nucleic acid can be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g. [00153] Proteins, vectors, plasmids, and the nucleic acid editing systems provided herein can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to construct any embodiment of the compositions provided herein are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. [00154] A nuclease, nucleic acid editing system, fusion protein, or a polynucleotide encoding a nuclease, nucleic acid editing system, fusion protein can be delivered to a cell by electroporation. Electroporation using, for example, the NEON® Transfection System (ThermoFisher Scientific) or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery of nucleic acids and proteins into a cell. For example, a nuclease or fusion protein provided herein can be purified and complexed with a suitable guide nucleic acid for delivery into a cell. Electroporation parameters can be adjusted to optimize delivery efficiency and/or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some embodiments, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type can be adjusted to optimize transfection efficiency and/or cell viability. [00155] The compositions provided herein can be delivered to any suitable cell. Suitable cells can include but are not limited to eukaryotic and prokaryotic cells and/or cell lines. A suitable cell can be a human primary cell. A primary cell can be taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. [00156] A primary cell can be acquired from a variety of sources such as an organ, vasculature, buffy coat, whole blood, apheresis, plasma, bone marrow, tumor, cell-bank, cryopreservation bank, or a blood sample. A primary cell can be a stem cell. A suitable cells that can be edited with a nucleic acid editing system provided herein include but are not limited to: epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B, NK, and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, Leydig cells, peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, dopaminergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, dopaminergic cells, embryonic stem cells, fibroblasts and fetal fibroblasts. Further, the one or more cells can be pancreatic islet cells and/or cell clusters or the like, including, but not limited to pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), or pancreatic ε cells. In one instance, the one or more cells can be pancreatic α cells. In another instance, the one or more cells can be pancreatic β cells. [00157] A human primary cell can be an immune cell. An immune cell can be a T cell, B cell, NK cell, and/or a macrophage. Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells. Suitable cells can comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells. Suitable cells can be progenitor cells. Suitable cells can be derived from the subject to be treated (e.g., a subject with a disease, a subject in need of treatment, or a subject that is immunocompromised). Suitable cells can be derived from a human donor. [00158] A method of attaining suitable cells, such as human primary cells, can comprise selecting cells. In some embodiments, a cell can comprise a marker that can be selected for the cell. For example, such marker can comprise GFP, a resistance gene (for example, a gene conferring antibiotic resistance), a cell surface marker, an endogenous tag. Cells can be selected using any endogenous marker. Suitable cells can be selected using any technology. Such technology can comprise flow cytometry and/or magnetic columns. The selected cells can then be infused into a subject. The selected cells can also be expanded to large numbers. The selected cells can be expanded prior to infusion. [00159] Delivery vehicles for in vivo and ex vivo use can include pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. 7. Pharmaceutical Compositions, Dosage, and Administration [00160] Provided herein is a pharmaceutical composition comprising a nuclease, nucleic acid editing system, or a fusion protein provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient. Further provided herein is a pharmaceutical composition comprising a vector provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient. [00161] In some embodiments, compositions provided herein (e.g., nucleic acid editing systems, nucleases, and fusion proteins provided herein) are combined with pharmaceutically acceptable salts, excipients, and/or carriers to form a pharmaceutical composition. Pharmaceutical salts, excipients, and carriers may be chosen based on the route of administration, the location of the target issue, and the time course of delivery of the drug. A pharmaceutically acceptable carrier or excipient may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. [00162] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol). Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. [00163] Compositions provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage. A dosage unit form is a physically discrete unit of a composition provided herein appropriate for a subject to be treated. For any composition provided herein the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human use. [00164] Provided herein are compositions and pharmaceutical compositions for administering to a subject in need thereof (e.g., as a treatment of a disease). In some embodiments, pharmaceutical compositions provided herein are in a form that allows for compositions provided herein to be administered to a subject. In some embodiments, the pharmaceutical composition is formulated for intratumoral delivery. In some embodiments, administration of a pharmaceutical composition provided herein is local administration or systemic administration. In some embodiments, a pharmaceutical composition provided herein is formulated for administration / for use in administration via an intratumoral, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, or intracranial route. In some embodiments, the administering is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours. In some embodiments, the administering is daily, weekly, or monthly. In some embodiments, the administering is repeated at least about every 28 days or 56 days. [00165] Vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual subject (e.g., lymphocytes, T cells, bone marrow aspirates, or tissue biopsy), followed by reimplantation of the cells into a subject, usually after selection for cells which have incorporated the vector. Prior to or after selection, the cells can be expanded. [00166] Provided herein are cells expressing a nuclease, fusion protein, or a nucleic acid editing system provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a nucleic acid encoding for a nuclease, fusion protein, or a nucleic acid editing system provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a vector encoding for an engineered protein provided here nuclease, fusion protein, or a nucleic acid editing system provided herein. [00167] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte, a leukocyte, a T cell, a natural killer cell, a macrophage, a neutrophil, an eosinophil, a basophil, a dendritic cell, a stem cell, a cancer cell, a stem cell, a heart cell, a pancreatic cell, a neuron, an induced pluripotent stem-cell derived cell (iPSC), or an in-vitro differentiated iPSC. In some embodiments, the cell is a genetically modified cell. In some embodiments, the genetically modified cell is a chimeric antigen receptor (CAR) T cell, an engineered T cell receptor (TCR) T cell, or a tumor-infiltrating lymphocyte (TIL). [00168] Cells provided herein can be administered to a subject in need thereof, e.g., a subject with a disease or a condition. Therapeutic applications are discussed further below. 8. Gene Editing Applications & Diagnostics [00169] Provided herein are methods of modifying a target nucleic acid. In some embodiments, the methods comprise, contacting a target nucleic acid molecule with a nuclease, fusion protein, nucleic acid editing system or composition provided herein. In some embodiments, the nuclease comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182. In some embodiments, the nucleic acid editing system provided herein comprises a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex. In some embodiments, the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule. [00170] In some embodiments, the percentage of target nucleic acid molecule modification is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. In some embodiments, the method of modifying the target nucleic acid further comprises mutation of a target nucleic acid sequence. In some embodiments, the mutation comprises an insertion or deletion at said one or more nucleic acid sequences. In some embodiments, the mutation comprises an indel. In some embodiments, the methods further comprise delivering the composition to a cell. In some embodiments, the methods further comprise delivering to the cell at least one exogenous nucleic acid molecule for insertion at the cleavage site. In some embodiments, the at least one exogenous nucleic acid molecule is delivered with a recombination polypeptide. In some embodiments, the recombination polypeptide is an integrase, a flippase, a transponase, or a recombinase. In some embodiments, the exogenous nucleic acid molecule comprises at least one sequence having at least 75% homology to the target nucleic acid molecule. In some embodiments, the method is in vitro or in vivo. [00171] Provided herein are methods of ex vivo modifying a cell. In some embodiments, the methods comprise: contacting a cell with a nuclease, nucleic acid editing system, fusion protein, or a composition provided herein under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell. [00172] Further provided herein are methods of ex vivo modifying a cell, the method comprising: contacting a cell with a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises: (i) a nuclease provided herein; and (ii) a guide RNA that binds to a target nucleic acid, wherein upon contacting the cell with the RNP complex, the nuclease cleaves a target nucleic acid molecule, thereby modifying said cell. In some embodiments, the cell is an immune cell or a stem cell. In some embodiments, the immune cell is a leukocyte, a lymphocyte, a natural killer cell, a dendritic cell, a macrophage, a myeloid cell, a T-cell, a B cell, a stem cell, an induced- pluripotent derived cell, a cancer cell, or an endothelial cell. In some embodiments, the stem cell is an embryonic stem cell, an induced-pluripotent stem cell (iPSC), or an adult stem cell. [00173] Methods of modifying a target nucleic acid provided herein can be used for agricultural applications, e.g, generation of improved plant species; biomedical applications; drug screening; and therapeutic treatments. [00174] Further provided herein are methods of detecting one or more target nucleic acids in a test sample, the method comprising: (a) immobilizing a guide nucleic acid onto a solid support; and (b) contacting the guide nucleic acid with: (i) a test sample; and (ii) a nuclease provided herein. In some embodiments the test sample comprises a target nucleic acid capable of binding to the guide nucleic acid. In some embodiments, a complex is formed between the guide nucleic acid, a nuclease provided herein, and the target nucleic acid. In some embodiments, upon formation of the complex the nuclease cleaves the target nucleic acid. In some embodiments, the method further comprises detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample. In some embodiments, the guide nucleic acid comprises a sequence of any one of: SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152. In some embodiments, the nuclease comprises a sequence set forth in any one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182. In some embodiments, prior to the detecting step, the method further comprises, amplifying the target nucleic acid. In some embodiments, the amplifying comprises polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), improved multiple displacement amplification (EVIDA), l simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), transcription mediated amplification (TMA), ramification amplification method (RAM), or any combination thereof. In some embodiments, the methods further comprise performing an endonuclease mismatch detection assay, an immunoassay, gel electrophoresis, a plasmid interference assay, nucleic acid sequencing, or any combination thereof. In some embodiments, the detecting comprises calorimetric detection, potentiometric detection, amperometric detection, optical detection, piezo-electric detection, or any combination thereof. [00175] In some embodiments, the solid support comprises a reaction chip, a paper, a quartz microfiber, mixed esters of cellulose, a porous aluminum oxide, a patterned surface, a tube, a well, or a matrix. n some embodiments, the solid support comprises a patterned surface suitable for immobilization of molecules in an ordered pattern. In some embodiments a patterned surface refers to an arrangement of different regions in or on an exposed layer of a solid support. In some embodiments, the solid support comprises an array of wells or depressions in a surface. The composition and geometry of the solid support can vary with its use. In some embodiments, the solid support is a planar structure such as a slide, chip, microchip and/or array. As such, the surface of the substrate can be in the form of a planar layer. In some embodiments, the solid support comprises one or more surfaces of a flowcell. A flowcell is a type of chamber comprising a solid surface across which one or more fluid reagents can be flowed. In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or vessel. In some embodiments, the solid support comprise microspheres or beads. Microspheres, beads, or particles can be made of various material including, but not limited to, plastics, ceramics, glass, and polystyrene. In some embodiments, the microspheres are magnetic microspheres or beads. Alternatively or additionally, the beads may be porous. The bead sizes range from nanometers, e.g., about 100 nm, to millimeters, e.g., about 1 mm. [00176] In some embodiments, the test sample comprises a liquid sample. In some embodiments, the test sample comprises a biological sample or an environmental sample. [00177] In some embodiments, the environmental sample comprises a food sample, a paper surface, a fabric, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a saline water sample, or any combination thereof. In some embodiments, the environmental sample is suspected of comprising a pathogenic microbial organism. Sequences specific to a pathogen of interest may be identified or selected by comparing the coding sequences from the pathogen of interest to all coding sequences in other organisms by BLAST software. [00178] Appropriate samples for use in the methods provided herein include any conventional biological sample obtained from an organism or a part thereof, such as a plant, animal, bacteria, and the like. In particular embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface. A sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. Exemplary samples include, without limitation, cells, cell lysates, blood smears, cytocentrifuge preparations, cytology smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine-needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections). In other examples, the sample includes circulating tumor cells (which can be identified by cell surface markers). In particular examples, samples are used directly (e.g., fresh or frozen), or can be manipulated prior to use, for example, by fixation (e.g., using formalin) and/or embedding in wax (such as formalin-fixed paraffin-embedded (FFPE) tissue samples). It will be appreciated that any method of obtaining tissue from a subject can be utilized, and that the selection of the method used will depend upon various factors such as the type of tissue, age of the subject, or procedures available to the practitioner. In some embodiments, the biological sample comprises a cell extract, cell medium, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a bodily secretion, a transudate, an exudate, a skin swab, or a mucosal membrane swab. In some embodiments, the test sample is from a subject that has, is suspected of having, or at risk of developing a disease or a disorder. [00179] The nucleic acid editing systems and compositions provided herein have a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. Further provided herein are methods of treating a disease, a disorder, or a condition. Non- limiting examples of diseases and conditions that can be treated using the compositions and methods provided herein include cancer, autoimmune diseases, diabetes, heart disease, infectious diseases, lower respiratory infections, neonatal conditions, neurological diseases, neuromuscular diseases, muscular diseases, gastrointestinal diseases, kidney diseases, liver diseases, vascular diseases, deafness, blindness, and the like. [00180] In some embodiments, the methods comprise detecting one or more target nucleic acids in a test sample obtained from a subject using the methods provided herein; identifying a subject as having a target nucleic acid; and providing an appropriate treatment for the disease, disorder, or condition. In some embodiments, the method comprises using a nuclease provided herein to cleave a diseased DNA or RNA in a cell. The disease DNA or RNA may yield translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a RNA transcribed from a gene that becomes expressed at an abnormally high level; it may be a RNA transcribed from a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. The disease- associated nucleic acid can include RNA transcribed from a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The translated products may be known or unknown, and may be at a normal or abnormal level. The target nucleic acid of a nucleic acid editing system provided herein can be any endogenous or exogenous nucleic acid of a eukaryotic cell. For example, the target nucleic acid can be a nucleic acid residing in the nucleus of the eukaryotic cell. The target nucleic acid can encode for a gene product (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA, tRNA, or rRNA). 9. Scaffolds and Systems [00181] Provided herein are scaffolds, wherein the scaffolds comprise: a nuclease, a guide nucleic acid, a fusion protein, or a nucleic acid editing system provided herein. In some embodiments, a scaffold comprises: a nuclease provided herein; and a guide nucleic acid provided herein, wherein the nuclease and/or the guide nucleic acid are immobilized to the scaffold. In some embodiments, the scaffold comprises a set of nucleases. In some embodiments, the set of nucleases comprise at least one nuclease comprising an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77-78, or 182, a functional fragment, or a derivative thereof. In some embodiments, the scaffold comprises a set of guide nucleic acids, wherein the set of guide nucleic acids comprise a direct repeat sequence of any one or more of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, at least one guide nucleic acid is immobilized to the scaffold. In some embodiments, the set of nucleases are immobilized to the scaffold. In some embodiments, at least one nuclease and at least one guide nucleic acid are immobilized to the scaffold. In some embodiments, at least one nuclease is immobilized to the scaffold and the at least one guide nucleic acid is in complex with the at least one nuclease. In some embodiments, the set of guide nucleic acids are each immobilized to the scaffold; and the at least one nuclease is in complex with at least one guide sequence. In some embodiments, at least one nuclease comprises an amino acid sequence with at least 90% identity to one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182. In some embodiments, at least one nuclease comprises an amino acid sequence with at least 95% identity to one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182. In some embodiments, at least one nuclease comprises an amino acid sequence of one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182. In some embodiments, the guide nucleic acid comprises a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6; and a variable sequence. In some embodiments, the variable sequence is at least 75% complementary to a target sequence. In some embodiments, the scaffold further comprises a target sequence in complex with the at least one nuclease and at least one guide nucleic acid. [00182] Further provided herein are systems comprising a scaffold provided herein. In some embodiments, the systems comprise: (a) a scaffold provided herein; (b) a reporter molecule; and (c) a detector. In some embodiments, when a target nucleic acid forms a complex with the scaffold, the reporter molecule produces a detectable signal that is detected by the detector. In some embodiments, the system further comprises reagents for nucleic acid amplification. In some embodiments, the reporter molecule is selected from the group consisting of: a fluorophore, a dye, a polypeptide, an antibody, a nucleic acid, and any combination thereof. In some embodiments, the detectable signal is a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. 10. Kits [00183] Provided herein are kits comprising a nuclease, a guide nucleic acid, a fusion protein, or a nucleic acid editing system provided herein. Further provided herein are kits, wherein the kits comprise: a gene editing system provided herein, packaging and materials therefor. [00184] In some embodiments, a formulation of a composition described herein is prepared in a single container for administration. In some embodiments, a formulation of a composition provided herein is prepared two containers for administration, separating the nucleic acids and/or the nucleases provided herein. As used herein, “container” includes vessel, vial, ampule, tube, cup, box, bottle, flask, jar, dish, well of a single-well or multi-well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and/or transported, and accessed to remove the contents. Examples of such containers include glass and/or plastic sealed or re-sealable tubes and ampules, including those having a rubber septum or other sealing means that is compatible with withdrawal of the contents using a needle and syringe. In some embodiments, the containers are RNase free. [00185] Further provided herein are kits, wherein the kits comprise: a first container comprising a nuclease provided herein; and a second container comprising a guide nucleic acid provided herein, packaging and materials therefor. 11. Exemplary Embodiments [00186] Provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 77, a functional fragment, or a derivative thereof. Further provided herein are nucleases, wherein the nucleases comprise an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182, a functional fragment, or a derivative thereof. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 78. In some embodiments, a nuclease provided herein comprises an amino acid sequence of SEQ ID NO: 182. Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 119, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe. Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe comprising a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 20; or SEQ ID NO: 21; and (b) a helical recognition (REC) lobe. Further provided herein are nucleases, wherein the nucleases comprise: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe. [00187] Provided herein are nucleic acid sequences, wherein the nucleic acid sequences encode for a nuclease provided herein. Further provided herein are ribonucleoprotein (RNP) complexes, wherein the RNP complexes comprises: (i) a nuclease provided herein; and (ii) a guide RNA that binds to a target nucleic acid. Further provided herein are vectors, wherein the vectors comprise a nucleic acid sequence encoding for a nuclease provided herein. Further provided herein are viral vectors, wherein the viral vectors comprise a nucleic acid sequence encoding for a nuclease provided herein. Further provided herein are nucleic acid editing systems comprising a nuclease provided herein. [00188] Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: a nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64 wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: a nuclease comprising (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing systems comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease or the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 5, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 2, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 11, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein the guide nucleic acid comprises a direct repeat sequence set forth in SEQ ID NO: 6, and wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid. Further provided herein are nucleic acid editing systems comprising: (a) a nuclease provided herein; and (b) a guide nucleic acid sequence comprising: a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6 operably linked to a variable sequence, wherein the variable sequence is complementary to a target nucleic acid. Further provided herein are nucleic acid editing systems, wherein upon formation of the complex, the nuclease generates a break in the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nuclease generates a single-stranded break in the target nucleic acid or a double-stranded break in the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the nuclease generates a double-stranded break with staggered 5’ overhangs in the target nucleic acid. Further provided herein are nucleic acid editing systems, wherein the target nucleic acid is a DNA. Further provided herein are nucleic acid editing systems, wherein the nuclease further comprises one or more nuclear localization sequence (NLS). Further provided herein are nucleic acid editing systems, wherein the one or more NLS is an SV40 Large NLS, a large T antigen NLS, a c-Myc NLS, a nucleoplasmin NLS, an EGL-13 NLS, or a TUS-protein NLS. Further provided herein are nucleic acid editing systems, wherein the NLS comprises an SV40 Large NLS, and wherein the SV40 Large NLS comprises an amino acid sequence of: MGPKKKRKV (SEQ ID NO: 3). Further provided herein are nucleic acid editing systems, wherein the NLS comprises a nucleoplasmin NLS, and wherein the nucleoplasmin NLS comprises an amino acid sequence of: KRPAATKKAGQAKKKK (SEQ ID NO: 4). Further provided herein are nucleic acid editing systems, wherein the nuclease further comprises: (a) an SV40 Large NLS; and (b) a nucleoplasmin NLS. Further provided herein are nucleic acid editing systems, wherein the nuclease further comprises: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 63, or SEQ ID NO: 153. [00189] Provided herein are nucleic acid editing systems, wherein the nuclease further comprises one or more peptide linkers. Further provided herein are nucleic acid editing systems, wherein the nuclease further comprises a C-terminal tag. Further provided herein are nucleic acid editing systems, wherein the C-terminal tag is a c-myc tag. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid comprises RNA. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid comprises RNA and at least one modified nucleoside. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid comprises one or more direct repeat sequences. Further provided herein are nucleic acid editing systems, wherein the one or more direct repeat sequences comprises an sequence of: SEQ ID NO: 5, SEQ ID NO: 6, a functional fragment thereof; or a sequence that is complementary to any one of SEQ ID NO: 5 or SEQ ID NO: 6, or a functional fragment thereof. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid further comprises a sequence that is at least partially complementary to the target nucleic acid sequence. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid comprises two direct repeat sequences and a variable sequence, wherein the variable sequence is at least 75% complementary to a target nucleic acid sequence. Further provided herein are nucleic acid editing systems, wherein the guide nucleic acid further comprises a promoter sequence. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 77. the nuclease comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78. Further provided herein are nucleic acid editing systems, wherein the nuclease comprises an amino acid sequence that is identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78. Further provided herein are nucleic acid editing systems, wherein the nucleic acid editing system is delivered inside a cell or a cellular organelle via electroporation, nucleofection, conjugation, lipofection, calcium phosphate precipitation, or a delivery vehicle. [00190] Provided herein are compositions, wherein the compositions comprise a delivery vehicle; and a nuclease provided herein or a polynucleotide sequence encoding for a nuclease provided herein. Further provided herein are compositions, wherein the compositions comprise: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof. Further provided herein are compositions, wherein the vector is a viral vector. Further provided herein are compositions, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an oncolytic viral vector. Further provided herein are compositions, wherein the compositions further comprise a guide nucleic acid. Further provided herein are compositions, wherein the guide nucleic acid comprises a sequence of any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152. Further provided herein are compositions, wherein the guide nucleic acid further comprises a sequence that is at least partially complementary to a target nucleic acid sequence. Further provided herein are compositions, wherein the guide nucleic acid comprises SEQ ID NO: 5 or SEQ ID NO: 6; and a sequence that is at least partially complementary to a target nucleic acid sequence. [00191] Provided herein are viral vectors, wherein the viral vectors comprise: a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof. Further provided herein are viral vectors, wherein the viral vectors comprise a nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NOS: 5-6. Further provided herein are viral vectors, wherein the viral vectors comprise: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid comprising any one of SEQ ID NOS: 5-9, 36-39, 53-60. Further provided herein are compositions, wherein the compositions comprise: a viral vector provided herein; and a delivery vehicle. Further provided herein are viral vectors and compositions, wherein the delivery vehicle is an emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, or a polymer. Further provided herein are viral vectors, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an oncolytic viral vector. Further provided herein are lentiviral vectors, wherein the lentiviral vectors comprise: a nuclease provided herein, a functional fragment, or a derivative thereof. Further provided herein are lentiviral vectors, wherein the lentiviral vectors comprise: (a) a nuclease provided herein, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid provided herein. [00192] Provided herein are kits, wherein the kits comprise a nuclease provided herein. Further provided herein are kits, wherein the kits comprise: a first container comprising a nuclease provided herein; and a second container comprising a guide nucleic acid provided herein, packaging and materials therefor. Further provided herein are kits, wherein the kits comprise: a gene editing system provided herein, packaging and materials therefor. [00193] Provided herein are methods of modifying a target nucleic acid molecule, wherein the methods comprise: contacting a target nucleic acid molecule with a composition comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182; and (b) a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex, wherein the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule. Further provided herein are methods, wherein the percentage of target nucleic acid molecule modification is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. Further provided herein are methods, wherein the methods further comprise mutation of a target nucleic acid sequence. Further provided herein are methods, wherein the mutation comprises an insertion or deletion at said one or more nucleic acid sequences. Further provided herein are methods, wherein the methods further comprise delivering the composition to a cell. Further provided herein are methods, wherein the methods further comprise delivering to the cell at least one exogenous nucleic acid molecule for insertion at the cleavage site. Further provided herein are methods, wherein the at least one exogenous nucleic acid molecule is delivered with a recombination polypeptide. In some embodiments, the recombination polypeptide is an integrase, a flippase, a transponase, or a recombinase. Further provided herein are methods, wherein the exogenous nucleic acid molecule comprises at least one sequence having at least 75% homology to the target nucleic acid molecule. Further provided herein are methods, wherein the methods are performed in vitro or in vivo. [00194] Provided herein are methods of ex vivo modifying a cell, wherein the methods comprise: contacting a cell with a composition provided herein under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell. Further provided herein are methods of ex vivo modifying a cell, wherein the methods comprise: contacting a cell with a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises: (i) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182; and (ii) a guide RNA that binds to a target nucleic acid, wherein upon contacting the cell with the RNP complex, the nuclease cleaves a target nucleic acid molecule, thereby modifying said cell. Further provided herein are methods, wherein the cell is an immune cell or a stem cell. Further provided herein are methods, wherein the immune cell is a leukocyte, a lymphocyte, a natural killer cell, a dendritic cell, a macrophage, a myeloid cell, a T-cell, a stem cell, an induced-pluripotent derived cell, a cancer cell, or an endothelial cell. Further provided herein are methods, wherein the stem cell is an embryonic stem cell, an induced-pluripotent stem cell (iPSC), or an adult stem cell. [00195] Provided herein are methods of detecting one or more target nucleic acid molecules in a test sample, wherein the methods comprise: (a) immobilizing a guide nucleic acid onto a solid support; and (b) contacting the guide nucleic acid with: (i) a test sample; and (ii) a nuclease comprising an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182, wherein when the test sample comprises a target nucleic acid capable of binding to the guide nucleic acid, a complex is formed between the guide nucleic acid, the nuclease, and the target nucleic acid, and wherein upon formation of the complex the nuclease cleaves the target nucleic acid; and (c) detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample. Further provided herein are methods, wherein the guide nucleic acid comprises a sequence of any one of: SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152, or a sequence that is complementary to any one of SEQ ID NOS: 5-9, 36-39, 53-60, 79-84, 113-152. wherein prior to step (c), the method further comprises, amplifying the target nucleic acid. Further provided herein are methods, wherein the amplifying comprises polymerase chain reaction (PCR), nucleic acid sequence- based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), improved multiple displacement amplification (EVIDA), l simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), transcription mediated amplification (TMA), ramification amplification method (RAM), or any combination thereof. Further provided herein are methods, wherein the methods further comprise performing an endonuclease mismatch detection assay, an immunoassay, gel electrophoresis, a plasmid interference assay, nucleic acid sequencing, or any combination thereof. Further provided herein are methods, wherein the detecting comprises calorimetric detection, potentiometric detection, amperometric detection, optical detection, piezo-electric detection, or any combination thereof. Further provided herein are methods, wherein the solid support comprises a reaction chip, a paper, a quartz microfiber, mixed esters of cellulose, a porous aluminum oxide, a patterned surface, a tube, a well, or a matrix. Further provided herein are methods, wherein the test sample comprises a liquid sample. Further provided herein are methods, wherein the test sample comprises a biological sample or an environmental sample. Further provided herein are methods, wherein the biological sample comprises a cell extract, cell medium, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a bodily secretion, a transudate, an exudate, a skin swab, or a mucosal membrane swab. Further provided herein are methods, wherein the environmental sample comprises a food sample, a paper surface, a fabric, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a saline water sample, or any combination thereof. Further provided herein are methods, wherein the test sample is from a subject that has or is suspected of having a disease or a disorder. Further provided herein are methods, wherein the target nucleic acid is DNA. [00196] Provided herein are uses for a nuclease provided herein. Further provided herein are uses for a nuclease provided herein, wherein the nuclease comprises: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe. Further provided herein are uses for a nuclease, wherein the NUC lobe further comprises: a wedge 1 (WED1) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 16; or SEQ ID NO: 17; and/or a WED 3 domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 18; or SEQ ID NO: 19. Further provided herein are uses for a nuclease, wherein the NUC lobe further comprises: a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 20; or SEQ ID NO: 21. Further provided herein are uses for a nuclease, wherein the NUC lobe further comprises: a RuvCI domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 22; or SEQ ID NO: 23. Further provided herein are uses for a nuclease, wherein the NUC lobe further comprises: a RuvCII domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 24; or SEQ ID NO: 25. Further provided herein are uses for a nuclease, wherein the NUC lobe further comprises a nuclease domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 26; or SEQ ID NO: 27. Further provided herein are uses for a nuclease, wherein the NUC lobe further comprises: a RuvCIII domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 28; or SEQ ID NO: 29. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 11, 2, 10, 11, 77, or 78. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are uses for a nuclease, wherein the nuclease comprises an amino acid sequence comprising one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. [00197] Provided herein are scaffolds comprising a nuclease or a gene editing system provided herein, wherein the nuclease or the gene editing system is immobilized to the scaffold. Further provided herein are scaffolds, wherein the scaffolds comprise: (a) a set of nucleases, wherein at least one nuclease comprises an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a set of guide nucleic acids, wherein the set of nucleases and/or the set of guide nucleic acids are immobilized to the scaffold. Further provided herein are scaffolds, wherein the set of nucleases comprise at least one nuclease comprising an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof. Further provided herein are scaffolds, wherein the scaffolds comprise a set of guide nucleic acids, wherein the set of guide nucleic acids comprise a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Further provided herein are scaffolds, wherein at least one guide nucleic acid is immobilized to the scaffold. Further provided herein are scaffolds, wherein the set of nucleases are immobilized to the scaffold. Further provided herein are scaffolds, wherein at least one nuclease and at least one guide nucleic acid are immobilized to the scaffold. Further provided herein are scaffolds, wherein at least one nuclease is immobilized to the scaffold and the at least one guide nucleic acid is in complex with the at least one nuclease. Further provided herein are scaffolds, wherein the set of guide nucleic acids are each immobilized to the scaffold; and the at least one nuclease is in complex with at least one guide sequence. Further provided herein are scaffolds, wherein at least one nuclease comprises an amino acid sequence with at least 90% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are scaffolds, wherein at least one nuclease comprises an amino acid sequence with at least 95% identity to one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are scaffolds, wherein at least one nuclease comprises an amino acid sequence of one of SEQ ID NOS: 1, 2, 10, 11, 77, or 78. Further provided herein are scaffolds, wherein the guide nucleic acid comprises a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6; and a variable sequence. Further provided herein are scaffolds, wherein the variable sequence is at least 75% complementary to a target sequence. Further provided herein are scaffolds, wherein the scaffold further comprises a target sequence in complex with the at least one nuclease and at least one guide nucleic acid. [00198] Provided herein are systems and devices comprising: (a) a scaffold provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when a target nucleic acid forms a complex with the scaffold, the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector. Further provided herein are systems and devices comprising: (a) a nuclease provided herein; (b) a reporter molecule or a phase changing agent; and (c) a detector, wherein, when the nuclease cleaves a target nucleic acid the reporter molecule or phase changing agent produces a detectable signal that is detected by the detector. the system further comprises reagents for nucleic acid amplification. Further provided herein are systems, wherein the reporter molecule is selected from the group consisting of: a fluorophore, a dye, a polypeptide, an antibody, a nucleic acid, and any combination thereof. Further provided herein are systems, wherein the detectable signal is a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. [00199] Provided herein are fusion proteins, wherein the fusion proteins comprise a nuclease provided herein. Further provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [00200] Provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [00201] Provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [00202] Provided herein are fusion proteins, wherein the fusion proteins comprise: (a) a first protein construct comprising: (a) a first protein construct comprising: a first nuclease consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 78; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains. [00203] Provided herein are isolated proteins, wherein the isolated proteins comprise: an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. Further provided herein are isolated proteins, wherein the isolated proteins comprise any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182. [00204] Provided herein are isolated nucleic acids, wherein the isolated nucleic acids comprise: a sequence that encodes a protein that comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof. Further provided herein are isolated nucleic acids, wherein the isolated nucleic acids comprise: a sequence that encodes a protein that comprises an amino acid sequence comprising any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a fragment, or a derivative thereof. [00205] Provided herein are engineered nucleic acids, wherein the engineered nucleic acids comprise: a sequence that is at least 85% identical to SEQ ID NO: 90 or SEQ ID NO: 183. Further provided herein are engineered nucleic acids, wherein the engineered nucleic acids comprise SEQ ID NO: 90 or SEQ ID NO: 183. EXAMPLES EXAMPLE 1: NOVEL GENE EDITING SYSTEMS. [00206] Cell-based targeting of B2M and CD274 was performed with Nuclease #1 comprising a sequence of SEQ ID NO: 1 and a Nuclease #2 comprising a sequence of SEQ ID NO: 2 according to guided silencing effects and homolog-specific gRNA cassettes are described herein. [00207] 164 proteins were identified to have homology to two known gene editing nuclease proteins. A phylogenetic tree was generated and the percent sequence identity was compared for all sequences ranging from 19% to 50% sequence identity to the known gene editing nuclease protein. [00208] Of the 164 proteins, Nuclease #1 and Nuclease #2 were generated by standard cloning techniques along with eight other test nuclease sequences. Nuclease #1 and Nuclease #2 were each modified to include an SV40 NLS and nucleoplasmin NLS sequence yielding SEQ ID NO: 10 and SEQ ID NO: 11. Co-factor RNAs were designed using CRISPick software yielding SEQ ID NOS: 5 and 6 for direct repeat sequences. Full length targeting sequences for CD274 and B2M were designed and incorporated into expression cassettes with an hU6 promoter (SEQ ID NO: 31) summarized in Table 6. Table 6. Full length gRNA Expression Cassettes.
Figure imgf000079_0001
[00209] RKO cells were cultured and transfected with DNA encoding Nuclease #1 and DNA encoding a gRNA targeting CD274 or B2M (FIG. 2A). As a negative control, RKO cells were also transfected with a Nuclease #1 in the absence of a suitable guide RNA (CNTRL Nuc, no guide). Additional test nucleases were also assayed (labeled A- H). Nuclease #1 successfully targeted CD274 in RKO cells (FIG. 2B-FIG 2C) resulting in CD274 negative cells (3.79) as compared with CNTRL Nuc, no guide (1.56). [00210] T7 endonuclease (T7EN1) assays were performed.200 ng of gDNA was used to amplify the genomic locus of CD274 covering the guide targeting sequence. 510 bp PCR amplicons were purified and used for T7 endonuclease assays at 10 ng/µL. Samples from the T7EN1 assay were separated by agarose gel electrophoresis to detect the DNA cleavage products. The expected cleavage products were 400 base pairs (bps) and 100 bps using CD274 Guide #1 with control nuclease-CNTRL Nuc. Nuclease #1 and Nuclease #2 produced the expected bands 400 base pair cleavage product (FIG.3). [00211] Genomic analysis of B2M and CD274 targeting was performed (FIG. 4A-4B). Nuclease #1 and Nuclease #2 both produced indels/mismatches as using the same CD274 guide sequence (SEQ ID NO: 35). Nuclease #1 produced 1.8% indels targeting CD274 (FIG.4A) and 0.4% indels targeting B2M (FIG.4B). EXAMPLE 2: GUIDE RNA OPTIMIZATION FOR NUCLEASE #1 AND NUCLEASE #2. [00212] Optimization of the guide nucleic acid sequence was performed using CRISPick software to improve CD274 targeting by Nuclease #1 (SEQ ID NO: 10) and Nuclease #2 (SEQ ID NO: 11). Different combinations of direct repeat sequences and variable sequences were assayed with their corresponding nucleases. The guide sequences are summarized in Table 7. Table 7. Additional gRNA constructs.
Figure imgf000080_0001
[00213] For Nuclease #1, targeting activity was improved to 54% using the combination of direct repeat sequence, SEQ ID NO: 5, and variable sequence, SEQ ID NO: 7 (SEQ ID NO: 54, FIG. 5). For Nuclease #2, targeting activity was increased to 58% with direct repeat sequence, SEQ ID NO: 6, and variable sequence, SEQ ID NO: 7 (SEQ ID NO: 58, FIG. 6) and to 38% with SEQ ID NO: 9, respectively (SEQ ID NO: 60, FIG. 6). The targeting activity protocol is provided in Example 3 below. EXAMPLE 3: FLOW CYTOMETRY NUCLEASE TARGETING ACTIVITY ASSAY. [00214] In Examples 1 and 2, the following protocol was used to determine targeting activity of Nuclease #1 (SEQ ID NO: 10) and Nuclease #2 (SEQ ID NO: 11). [00215] Day 1 – Seeding of 600,000 RKO cells (CRL-2577, ATCC number) in 6-wells. Transfection of endonuclease and guide plasmids (1000 ng) after RKO cell attachment via Lipofection (LIPOFECTAMINE™ 3000). [00216] Day 2 – Medium exchange to 1 µg/mL puromycin for selection (DMEM (Gibco™), 10% FCS, no antibiotics). [00217] Day 3 – Medium exchange back to normal growth medium. [00218] Day 4 – Recovery from puromycin selection. [00219] Day 5 – Recovery from puromycin selection. [00220] Day 6 – Harvesting (trypsin-EDTA detachment) of puromycin-selected RKO cells for flow cytometric analysis. Antibody stainings of CD274 (AF647/APC) at dilutions of 1:20 in 2% FCS-PBS buffer. FACS analysis using a BD LSR Fortessa® cytometer. EXAMPLE 4: ADDITIONAL NUCLEASE AND GRNA COMPOSITIONS. [00221] Additional guides for B2M, CD46, CD81 and CD274 were designed and generated using CRISPick online tool for CRISPR KO guides. Delivery of guides via lentiviral transduction is performed including puromycin selection (PuroR) and eGFP pre-gating. [00222] Nuclease #1 and Nuclease #2 were transferred into a suitable lentiviral vector including blasticidin selection (BlastR). Suitable mutations and protein engineering of Nuclease #1 and Nuclease #2 are performed to enhance stability and cleavage efficiency of target DNA. Up to 50 different Nuclease #1 and Nuclease #2 mutants are designed and tested in RKO cells using the protocol outlined in Example 3. Lentiviral vectors comprising the Nuclease #1 or Nuclease #2 mutants are used to establish a pool of 50 to 100 nucleases for a high-throughput screening platform. [00223] Nuclease mutants are generated that are fully or partially nuclease-deficient. For nuclease #1, an Arginine at position 1165 can be substituted with an Alanine (R1165A) to produce a nuclease-deficient enzyme with SEQ ID NO: 61. Similarly, a nuclease-deficient mutant of Nuclease #2 is generated by a R1186A substitution as set forth in SEQ ID NO: 62. EXAMPLE 5: STRUCTURE-GUIDED ENGINEERING OF A NUCLEASE. [00224] The structure of the wild-type (CNTRL) nuclease was modeled using protein 3D modeling software. Amino acid substitutions were identified that would allow the nuclease to bind a DNA double helix and create a double strand break in a target sequence. Three mutants were identified as set out in Table 8. Table 8. REC and WED2 Mutant Nuclease Sequences.
Figure imgf000082_0001
[00225] Structure-guided mutagenesis was performed to generate nucleases with the amino acid substitutions in Table 8. A plasmid encoding the control and mutant nucleases were transfected into RKO cells with a guide RNA targeting (1) CD274, (2) CD46, or (3) B2M. Single stranded guide sequences targeting CD247, CD46, and B2M were generated and optimized as shown in Table 9. Table 9. Guide Sequence Target Sites.
Figure imgf000082_0002
[00226] Flow cytometry and T7 endonuclease assays were performed (FIG. 7A-7B, FIG. 8, and FIG. 9). Flow cytometry of RKO cells transfected with a mutant nuclease or the WT control nuclease targeting CD274 is shown in FIG. 8A. Double mutant #3 and the triple mutant exhibited a 2-fold reduction in the percentage of CD274 negative RKO cells (approx 40% CD274 negative) relative to the control nuclease with the same guide sequence (FIG. 7A). A T7 endonuclease assay confirmed that the mutant nucleases effectively disrupted the CD274 gene (FIG.7B). [00227] Flow cytometry of RKO cells transfected with a mutant nuclease or the WT control nuclease targeting CD46 is shown in FIG. 8. The WT control nuclease did not disrupt expression of CD46 in RKO cells. However, the mutant nucleases comprising single, double or triple mutants effectively reduced the percentage of CD46 RKO cells. Especially, the single mutant #1, the double mutant #2 and the triple mutant resulted in enhacned targeting of 40% CD46 negative RKO cells. Similarly, RKO cells transfected with the WT control nuclease and guide RNA targeting B2M did not disrupt the expression of B2M in RKO cells (FIG. 9). However, the mutant nucleases effectively disrupted B2M expression in RKO cells leading to enhanced targeting of approx. 30% using the triple mutant nuclease. Importantly, the accumulation of mutations led to additive targeting effects. EXAMPLE 6: NUCLEASE-SPECIFIC NUCLEAR LOCALIZATION SEQUENCE CONFIGURATIONS. [00228] Three nuclease localization sequence (NLS) configurations were generated and tested for delivery of a nuclease construct provided herein (FIGS.10A-10C). The configurations are provided in Table 10 and FIG.10A. Table 10. NLS Configuration Sequences.
Figure imgf000083_0001
Figure imgf000084_0001
[00229] Configuration sequences A-C increased CD247 targeting of the control nuclease by over 30% (FIG.10B-10C) and 1.36-fold higher relative to the initial sequence, Configuration 1. [00230] When the triple mutant nuclease (E155R, S532R, K538R) was delivered using configuration A in combination with a CD247-targeting guide sequence, targeting of CD247 was greater than 70% (FIG. 11A). This was also the case for targeting of B2M (FIG. 11B) and CD46 genomic loci (FIG.11C). [00231] RKO cells were co-transfected with the WT (SEQ ID NO: 85) or Triple Mutant nucleases (SEQ ID NO: 89 and 90) and sgRNAs targeting either B2M, CD274 or CD46. Genomic DNA was extracted from transfected RKO cells and genomic loci of B2M, CD274 and CD46 genes were PCR amplified. Next generation sequencing after polymerase chain reaction (PCR) amplification of genomic targets was performed to analyze and quantify genomic indel formation. NGS data derived from a MiSeq PE150 run was analyzed in order to identify and quantify the indel formation as shown in Table 11 and 12. The most dominant genomic targeting patterns have been summarized including an indel-specific quantification of editing frequencies. In summary, total targeting activities were quantified across all three genomic target sites B2M, CD46 and CD274 indicating editing efficiencies of >70% (FIG.12). [00232] NGS-derived cleavage patterns were determined following triple mutant transfection with CD274 or CD46 guide sequences as shown below in Tables 11 and Table 12. Table 11. CD274 cleavage patterns.
Figure imgf000084_0002
Figure imgf000085_0001
Table 12. CD46 cleavage patterns.
Figure imgf000085_0002
EXAMPLE 7: PROTOSPACER ADJACENT MOTIF (PAM) OPTIMIZATION AND GUIDE DESIGN. [00233] Twenty different guide sequences were designed that target sequences within the CD274 gene. Guides and protospacer adjacent motif (PAM) sequences are shown in Table 13 below. Table 13. PAM and Guide Sequences for CD274.
Figure imgf000085_0003
Figure imgf000086_0001
[00234] RKO cells were transfected with one of the guide sequences targeting CD274 and the Triple Mutant Nuclease or the WT control nuclease. Flow cytometry was performed as previously described. [00235] The Triple Mutant Nuclease (SEQ ID NO: 90) efficiently targeted TTNN PAMs and NTTN PAMs, indicating that the Triple Mutant Nuclease had greater flexibility for generating double strand breaks in the CD274 gene over the WT control nuclease (SEQ ID NO: 86). Essentially, the baseline targeting activity of the CD274 guide using a canonical TTTC PAM was used as a reference to rank alternative, non-canonical guides-PAM combinations. The WT control nuclease was inactive using the alternative guide-PAM combinations as only the TTCC PAM (TTNN motif) showed some minor targeting activity. In contrast, guide-PAM combinations of TTTN, TTNN and NTTN motifs resulted in robust targeting of CD274 by the triple mutant nuclease (FIG.13A and FIG.13B). EXAMPLE 8: Drop Out Fitness Screen of Engineered Nucleases for PAM Site Flexibility. [00236] A dropout fitness screen was conducted to explore the PAM site flexibility of the engineered triple mutant endonuclease at larger scale. RKO cells expressing the engineered triple mutant endonuclease (E155R/S532R/K538R) in conjunction with the optimized NLS configuration and a 2A-mCherry reporter (SEQ ID NO: 182 and SEQ ID NO: 183) were used for the fitness screen and guide RNA library transduction. The library of 5,994 guide RNAs expressed next to GFP and an antibiotic resistance cassette was designed to target 300 essential and 33 non-essential control genes covering a total of 18 different PAM sites for each target gene (SEQ ID NO: 184 and SEQ ID NO: 185). The guide RNA library was introduced via lentiviral transduction at a multiplicity of infection (MOI) of 0.1 (10% GFP-positive RKO cells) to integrate single guide RNAs into endonuclease expressing RKO cells. Antibiotic selection was employed for seven days to gain approximately 98% GFP-positive RKO cells expressing the endonuclease and a single guide RNA. [00237] Fitness effects relative to the starting point on day 0 (guide RNA library representation) were assessed on day 7 (end of antibiotic selection) and on day 21 (final time point) post guide RNA transduction (FIGS. 14A-14B). For this, guide RNA sequences integrated into the gDNA of RKO cells were PCR-amplified and evaluated via NGS. Counting of total guide RNAs enabled identification of the most active PAM - guide RNA combinations targeting essential genes as those combinations dropped out resulting in low guide counts (FIG. 14B). The dropout effects improved along the fitness screen as log fold-change (LFC) values were increased on day 21 with PAM sites “CTTC”, “TTTC” and “TTCC” identified as ideal candidates (FIG. 14B). Moreover, three additional PAM sites were identified which can be categorized as “NTTN” (ATTC, ATTG, GTTC). Of note, PAM motifs predicted to be inactive and summarized as "NTTT" as well as "TTTT" (i.e., ATTT, GTTT, TTTT) served as internal negative controls as those combinations did not drop out resulting in relatively high guide counts (positive LFCs, FIG.14B). [00238] The results of this dropout fitness screen confirm a gain in PAM site flexibility of the engineered triple mutant endonuclease which aligns with the arrayed PAM site screen data (FIGS. 13A-13B). Finally, both PAM site analyses allow to nominate three ideal (six possible) PAM sites for future guide RNA design using the engineered triple mutant endonuclease for gene editing applications. [00239] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
SEQUENCES
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
[00240] The following sequences (SEQ ID NOS: 32-35) are formatted to signify the promoter region, the direct repeat (DR) sequence, and variable sequence. Underlined signifies the DR and bold text signifies the variable sequence. Unformatted text is the promoter region of the expression cassette.
Figure imgf000093_0002
Docket No.199827-761601/PCT
Figure imgf000094_0001
[00241] The following sequences (SEQ ID NOS: 38-39) are formatted to signify the direct repeat (DR) sequence and variable sequence. Bold text signifies the variable sequence.
Figure imgf000094_0002
[00242] The following sequences (SEQ ID NO: 53-60) are formatted to signify the direct repeat (DR) sequence and variable sequence. Bold text signifies the variable sequence.
Figure imgf000094_0003
SEQ ID NO: 54: Nuclease #1 direct repeat with CD274-targeting guide RNA (bold: Variable Sequence)
Figure imgf000095_0001
[00243] The following sequences (SEQ ID NOS: 61-62) are formatted to signify the mutation that generates nickase activity. Underlined and bold text signifies the amino acid substitution as compared with reference sequences, SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
Figure imgf000095_0002
Figure imgf000096_0001
[00244] The following sequences (SEQ ID NOS: 64-78) are formatted to signify the the substitution made in the sequence relative to the reference sequence (SEQ ID NO: 1, or SEQ ID NOS: 12-15).
Figure imgf000096_0002
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
[00245] The following sequence (SEQ ID NO: 182) is formatted to signify the the amino acid substitutions made in the sequence relative to a reference sequence (e.g., SEQ ID NO: 1). Italicized and bold text signifies the 2A-self-cleaving peptide sequence (SEQ ID NO: 168). Capital letters indicate the endonuclease sequence. SEQ ID NO: 182: E155R/S532R/K538R Triple Mutant Nuclease – P2A (italicized and bold) – mCherry amino acid sequence
Figure imgf000110_0001
Figure imgf000111_0001
[00246] The following sequence (SEQ ID NO: 184) is formatted to signify the the nucleic acid substitutions made in the sequence relative to a reference sequence. SEQ ID NO: 184: DNA sequence encoding the guide RNA library scaffold with a GFP - P2A - PuroR - WPRE - hU6 - Optimized direct repeat (DR) (20 bp, bold italics) - Example guide (23 bp)
Figure imgf000112_0001
[00247] The following sequence (SEQ ID NO: 185) is formatted to signify the the amino acid substitutions made in the sequence relative to a reference sequence. SEQ ID NO: 185: GFP - P2A (italics)– PuroR (bold and italics) amino acid sequence of the guide RNA library vector.
Figure imgf000113_0001

Claims

CLAIMS What is claimed is: 1. A nucleic acid editing system, comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 77, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said nucleic acid editing system to said target nucleic acid, the nucleic acid editing system cleaves the target nucleic acid.
2. The nucleic acid editing system of claim 1, wherein the nucleic editing system forms a complex with the target nucleic acid, and wherein upon formation of the complex, the nuclease generates a break in the target nucleic acid.
3. The nucleic acid editing system of claim 2, wherein the nuclease generates a single- stranded break in the target nucleic acid or a double-stranded break in the target nucleic acid.
4. The nucleic acid editing system of claim 2, wherein the nuclease generates a double- stranded break with staggered 5’ overhangs in the target nucleic acid.
5. The nucleic acid editing system of claim 1, wherein the target nucleic acid is a DNA.
6. The nucleic acid editing system of claim 1, wherein the nuclease further comprises one or more nuclear localization sequence (NLS).
7. The nucleic acid editing system of claim 6, wherein the one or more NLS is an SV40 Large NLS, a large T antigen NLS, a c-Myc NLS, a nucleoplasmin NLS, an EGL-13 NLS, or a TUS-protein NLS.
8. The nucleic acid editing system of claim 7, wherein the NLS comprises an SV40 Large NLS, and wherein the SV40 Large NLS comprises an amino acid sequence of: MGPKKKRKV (SEQ ID NO: 3).
9. The nucleic acid editing system of claim 7, wherein the NLS comprises a nucleoplasmin NLS, and wherein the nucleoplasmin NLS comprises an amino acid sequence of: KRPAATKKAGQAKKKK (SEQ ID NO: 4).
10. The nucleic acid editing system of claim 7, wherein the NLS comprises a c-Myc NLS, and wherein the c-Myc NLS comprises an amino acid sequence of: MGPAAKRVKLDGSGS (SEQ ID NO: 153).
11. The nucleic acid editing system of claim 1, wherein the nuclease further comprises: (a) an SV40 Large NLS; (b) a nucleoplasmin NLS; (c) a c-Myc NLS; or (d) a combination of any one of (a)-(c).
12. The nucleic acid editing system of any one of claims 1 to 11, wherein the nuclease further comprises: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51, SEQ ID NO: 63, or SEQ ID NO: 153.
13. The nucleic acid editing system of claim 1, wherein the nuclease further comprises one or more peptide linkers.
14. The nucleic acid editing system of claim 1, wherein the nuclease further comprises a C-terminal tag.
15. The nucleic acid editing system of claim 14, wherein the C-terminal tag is a c-myc tag.
16. The nucleic acid editing system of claim 1, wherein the guide nucleic acid comprises RNA.
17. The nucleic acid editing system of claim 1, wherein the guide nucleic acid comprises RNA and at least one modified nucleoside.
18. The nucleic acid editing system of claim 1, wherein the guide nucleic acid comprises one or more direct repeat sequences.
19. The nucleic acid editing system of claim 16, wherein the one or more direct repeat sequences comprises an sequence of: SEQ ID NO: 5, SEQ ID NO: 6, a functional fragment thereof; or a sequence that is complementary to SEQ ID NO: 5, SEQ ID NO: 6, or a functional fragment thereof.
20. The nucleic acid editing system of claim 1, wherein the guide nucleic acid further comprises a sequence that is partially complementary to the target nucleic acid sequence.
21. The nucleic acid editing system of claim 16, wherein the guide nucleic acid comprises a direct repeat sequence and a variable sequence, wherein the variable sequence is at least 75% complementary to a target nucleic acid sequence.
22. The nucleic acid editing system of claim 1, wherein the guide nucleic acid further comprises a promoter sequence.
23. The nucleic acid editing system of claim 1, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of any one of SEQ ID NOS: 1, 2, or 77.
24. The nucleic acid editing system of claim 1, wherein the nuclease comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID NOS: 1 , 2, or 77.
25. The nucleic acid editing system of claim 1, wherein the nuclease comprises an amino acid sequence that is identical to SEQ ID NOS: 1 , 2, or 77.
26. A nucleic acid editing system, comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, SEQ ID NO: 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid that binds to a target nucleic acid, wherein upon binding of said guide nucleic acid to said target nucleic acid, the nuclease, the functional fragment, or the derivative thereof; the guide nucleic acid; and the target nucleic acid form a complex.
27. The nucleic acid editing system of claim 26, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182.
28. The nucleic acid editing system of claim 26, wherein the nuclease comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182.
29. The nucleic acid editing system of claim 26, wherein the nuclease comprises an amino acid sequence that is identical to an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 78, or SEQ ID NO: 182.
30. The nucleic acid editing system of any one of claims 1 to 29, wherein the nucleic acid editing system is delivered inside a cell or a cellular organelle via electroporation, nucleofection, conjugation, lipofection, calcium phosphate precipitation, or a delivery vehicle.
31. A composition comprising: (a) a delivery vehicle; and (b) a nucleic acid encoding for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof.
32. The composition of claim 31, wherein the delivery vehicle is an emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, or a polymer.
33. The composition of claim 32, wherein the vector is a viral vector.
34. The compositions of claim 33, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an oncolytic viral vector.
35. The composition of any one of claims 31 to 34, wherein the composition further comprises a guide nucleic acid.
36. The composition of claim 35, wherein the guide nucleic acid comprises a sequence of SEQ ID NO: 5, SEQ ID NO: 6, or a sequence that is complementary to SEQ ID NO: 5 or SEQ ID NO: 6.
37. The composition of claim 36, wherein the guide nucleic acid further comprises a sequence that is partially complementary to a target nucleic acid sequence.
38. The composition of claim 37, wherein the guide nucleic acid comprises SEQ ID NO: 5 or SEQ ID NO: 6; and a sequence that is partially complementary to a target nucleic acid sequence.
39. A viral vector comprising: a nucleic acid encoding for an amino acid sequence that is at least 85% identical to one of: SEQ ID NOS: 1, 2, 10, 11, 77-78, 182 a functional fragment, or a derivative thereof.
40. A viral vector comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a guide nucleic acid.
41. The viral vector of claim 39 or claim 40, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an oncolytic viral vector.
42. A kit comprising the nucleic acid editing system of any one of claims 1 to 30 or the composition of claim 31; and packaging materials therefor.
43. A method of modifying a target nucleic acid molecule, the method comprising: contacting a target nucleic acid molecule with a composition comprising: (a) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of one of: SEQ ID NOS: 1, 2, 10, 11, 64-78 or 182; and (b) a guide nucleic acid comprising a sequence that binds to the target nucleic acid, wherein upon binding of said guide nucleic acid to the target nucleic acid molecule, the nuclease, the guide nucleic acid, and the target nucleic acid molecule form a complex, and wherein the nuclease cleaves a target nucleic acid molecule generating a cleavage site within the target nucleic acid molecule, thereby modifying the target nucleic acid molecule.
44. The method of claim 43, wherein the percentage of target nucleic acid molecule modification is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
45. The method of claim 43 or claim 44, wherein modifying the target nucleic acid further comprises mutation of a target nucleic acid sequence.
46. The method of claim 45, wherein the mutation comprises an insertion or deletion at said one or more nucleic acid sequences.
47. The method of any one of claims 43 to 46, further comprising delivering the composition to a cell.
48. The method of claim 43, wherein the method further comprises delivering to the cell at least one exogenous nucleic acid molecule for insertion at the cleavage site.
49. The method of claim 48, wherein the at least one exogenous nucleic acid molecule is delivered with a recombination polypeptide.
50. The method of claim 49, wherein the recombination polypeptide is an integrase, a flippase, a transponase, or a recombinase.
51. The method of any one of claims 48 to 50, wherein the exogenous nucleic acid molecule comprises at least one sequence having at least 75% homology to the target nucleic acid molecule.
52. The method of any one of claims 43 to 51, wherein the method is in vitro or in vivo.
53. A method of ex vivo modifying a cell, the method comprising: contacting a cell with a composition of any one of claims 31 to 38 under conditions that permit nuclease cleavage of a target nucleic acid molecule, thereby modifying said cell.
54. A method of ex vivo modifying a cell, the method comprising: contacting a cell with a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises: (i) a nuclease comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78 or 182; and (ii) a guide RNA that binds to a target nucleic acid, wherein upon contacting the cell with the RNP complex, the nuclease cleaves a target nucleic acid molecule, thereby modifying said cell.
55. The method of claim 54, wherein the cell is an immune cell or a stem cell.
56. The method of claim 55, wherein the immune cell is a leukocyte, a lymphocyte, a natural killer cell, a dendritic cell, a macrophage, a myeloid cell, a T-cell, a stem cell, an induced-pluripotent derived cell, a cancer cell, or an endothelial cell.
57. The method of claim 55, wherein the stem cell is an embryonic stem cell, an induced- pluripotent stem cell (iPSC), or an adult stem cell.
58. A method of detecting one or more target nucleic acid molecules in a test sample, the method comprising: (a) immobilizing a guide nucleic acid onto a solid support; and (b) contacting the guide nucleic acid with: (i) a test sample; and (ii) a nuclease comprising an amino acid sequence that is at least 85% identical to any one of: SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182, wherein when the test sample comprises a target nucleic acid capable of binding to the guide nucleic acid, a complex is formed between the guide nucleic acid, the nuclease, and the target nucleic acid, and wherein upon formation of the complex the nuclease cleaves the target nucleic acid; and (c) detecting a signal indicating cleavage of the target nucleic acid molecule, thereby detecting the target nucleic acid in the sample.
59. The method of claim 58, wherein the guide nucleic acid comprises a sequence of SEQ ID NO: 5, SEQ ID NO: 6, or a sequence that is complementary to SEQ ID NO: 5 or SEQ ID NO: 6.
60. The method of claim 58 or claim 59, wherein prior to step (c), the method further comprises, amplifying the target nucleic acid.
61. The method of claim 60, wherein the amplifying comprises polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), improved multiple displacement amplification (EVIDA), l simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), transcription mediated amplification (TMA), ramification amplification method (RAM), or any combination thereof.
62. The method of any one of claims 58 to 61, further comprising performing an endonuclease mismatch detection assay, an immunoassay, gel electrophoresis, a plasmid interference assay, nucleic acid sequencing, or any combination thereof.
63. The method of any one of claims 58 to 62, wherein the detecting comprises calorimetric detection, potentiometric detection, amperometric detection, optical detection, piezo-electric detection, or any combination thereof.
64. The method of any one of claims 58 to 63, wherein the solid support comprises a reaction chip, a paper, a quartz microfiber, mixed esters of cellulose, a porous aluminum oxide, a patterned surface, a tube, a well, or a matrix.
65. The method of any one of claims 58 to 64, wherein the test sample comprises a liquid sample.
66. The method of any one of claims 58 to 65, wherein the test sample comprises a biological sample or an environmental sample.
67. The method of claim 66, wherein the biological sample comprises a cell extract, cell medium, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a bodily secretion, a transudate, an exudate, a skin swab, or a mucosal membrane swab.
68. The method of claim 67, wherein the environmental sample comprises a food sample, a paper surface, a fabric, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a saline water sample, or any combination thereof.
69. The method of any one of claims 58 to 68, wherein the test sample is from a subject that has or is suspected of having a disease or a disorder.
70. The method of any one of claims 58 to 69, wherein the target nucleic acid is DNA.
71. Use of a nuclease for cleaving a target nucleic acid, wherein the nuclease comprises: (a) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (b) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to the target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe.
72. Use of the nuclease of claim 71, wherein the NUC lobe further comprises: a wedge 1 (WED1) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 16 or SEQ ID NO: 17; and/or a WED 3 domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19.
73. Use of the nuclease of claim 71 or claim 72, wherein the NUC lobe further comprises: a Protospacer Adjacent Motif (PAM) Interacting (PI) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21.
74. Use of the nuclease of any one of claims 71 to 73, wherein the NUC lobe further comprises: a RuvCI domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 22 or SEQ ID NO: 23.
75. Use of the nuclease of any one of claims 71 to 74, wherein the NUC lobe further comprises: a RuvCII domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 24 or SEQ ID NO: 25.
76. Use of the nuclease any one of claims 71 to 75, wherein the NUC lobe further comprises a nuclease domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 26 or SEQ ID NO: 27.
77. Use of the nuclease any one of claims 71 to 76, wherein the NUC lobe further comprises: a RuvCIII domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 28 or SEQ ID NO: 29.
78. Use of the nuclease of any one of claims 71 to 77, wherein the nuclease comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 64- 78, or 182.
79. Use of the nuclease of any one of claims 71 to 78, wherein the nuclease comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 64- 78, or 182.
80. Use of the nuclease of any one of claims 71 to 79, wherein the nuclease comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 64- 78, or 182.
81. Use of the nuclease of any one of claims 71 to 80, wherein the nuclease comprises an amino acid sequence comprising one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182.
82. A scaffold comprising: (a) a set of nucleases, wherein at least one nuclease comprises an amino acid sequence comprising at least 85% identity to one of SEQ ID NOS: 1, 2, 10, 11, 64-78, 182, a functional fragment, or a derivative thereof; and (b) a set of guide nucleic acids, wherein the set of nucleases and/or the set of guide nucleic acids are immobilized to the scaffold.
83. The scaffold of claim 82, wherein at least one guide nucleic acid is immobilized to the scaffold.
84. The scaffold of claim 82, wherein the set of nucleases are immobilized to the scaffold.
85. The scaffold of any one of claims 82 to 84, wherein at least one nuclease and at least one guide nucleic acid are immobilized to the scaffold.
86. The scaffold of any one of claims 82 to 85, wherein at least one nuclease is immobilized to the scaffold and the at least one guide nucleic acid is in complex with the at least one nuclease.
87. The scaffold of any one of claims 82 to 85, wherein the set of guide nucleic acids are each immobilized to the scaffold; and the at least one nuclease is in complex with at least one guide sequence.
88. The scaffold of claim 82, wherein the at least one nuclease comprises an amino acid sequence with at least 90% identity to one of SEQ ID NOS: 1, 2, 10, 11, 64-78, or 182.
89. The scaffold of claim 82, wherein the at least one nuclease comprises an amino acid sequence with at least 95% identity to one of SEQ ID NOS: 1, 2, 10, 11, 64-78 or 182.
90. The scaffold of claim 82, wherein the at least one nuclease comprises an amino acid sequence of one of SEQ ID NOS: 1, 2, 10, 11, 64-78 or 182.
91. The scaffold of any one of claims 82 to 90, wherein the guide nucleic acid comprises a direct repeat sequence of SEQ ID NO: 5 or SEQ ID NO: 6; and a variable sequence.
92. The scaffold of claim 91, wherein the variable sequence is at least 75% complementary to a target sequence.
93. The scaffold of any one of claims 82 to 92, further comprising a target sequence in complex with the at least one nuclease and at least one guide nucleic acid.
94. A system comprising: (a) the scaffold of any one of claims 82 to 93; (b) a reporter molecule; and (c) a detector, wherein, when a target nucleic acid forms a complex with the scaffold, the reporter molecule produces a detectable signal that is detected by the detector.
95. The system of claim 94, wherein the system further comprises reagents for nucleic acid amplification.
96. The system of claim 94 or claim 95, wherein the reporter molecule is selected from the group consisting of: a fluorophore, a dye, a polypeptide, an antibody, a nucleic acid, and any combination thereof.
97. The system of any one of claims 94 to 96, wherein the detectable signal is a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo- electric signal.
98. A fusion protein comprising: (a) a first protein construct comprising: a first nuclease comprising a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains.
99. A fusion protein comprising: (a) a first protein construct comprising: a first nuclease comprising: (i) a nuclease (NUC) lobe, wherein the NUC lobe comprises at least one wedge 2 (WED2) domain comprising an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 66, SEQ ID NO: 68, or SEQ ID NO: 74; and (ii) a helical recognition (REC) lobe, wherein the REC lobe comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of: SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 64, wherein the nuclease is capable of binding to a target nucleic acid via the REC lobe in the presence of a guide nucleic acid, and wherein the nuclease capable of cleaving the target nucleic acid via the NUC lobe; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains.
100. A fusion protein comprising: (a) a first protein construct comprising: a nuclease comprising an amino acid sequence that is at least 85% identical to one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, 182, a functional fragment, or a derivative thereof; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains.
101. A fusion protein comprising: (a) a first protein construct comprising: a first nuclease consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 77, or SEQ ID NO: 78; and (b) a second protein construct comprising: a second nuclease, an acetylase, an acetyltransferase, an ATPase, an Argonaute protein, a base editor, a Cas polypeptide, a catalytically dead Cas polypeptide, a deacetylase, a deaminase, a decapping protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a transcriptional activator, a transcription activator-like effector nuclease (TALEN), a transcriptional repressor, a transposase, or a zinc finger, wherein the fusion protein optionally, comprises a linker between the first and second protein domains.
102. An isolated protein comprising an amino acid sequence that comprises at sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182.
103. An isolated protein comprising an amino acid sequence that comprises any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182.
104. An isolated nucleic acid comprising a sequence that encodes a protein that comprises a sequence that is at least 85% identical to any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182.
105. An isolated nucleic acid comprising a sequence that encodes a protein that comprises any one of SEQ ID NOS: 1, 2, 10, 11, 77, 78, or 182.
106. An engineered nucleic acid comprising a sequence that is at least 85% identical to SEQ ID NO: 90 or SEQ ID NO: 183.
107. An engineered nucleic acid comprising a sequence that comprises SEQ ID NO: 90 or SEQ ID NO: 183.
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