WO2020168291A1 - Enzymes with ruvc domains - Google Patents

Enzymes with ruvc domains Download PDF

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Publication number
WO2020168291A1
WO2020168291A1 PCT/US2020/018432 US2020018432W WO2020168291A1 WO 2020168291 A1 WO2020168291 A1 WO 2020168291A1 US 2020018432 W US2020018432 W US 2020018432W WO 2020168291 A1 WO2020168291 A1 WO 2020168291A1
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WIPO (PCT)
Prior art keywords
seq
sequence
endonuclease
nos
nuclease system
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US2020/018432
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English (en)
French (fr)
Inventor
Brian Thomas
Christopher Brown
Rose KANTOR
Audra DEVOTO
Cristina Butterfield
Lisa ALEXANDER
Daniela S. A. GOLTSMAN
Jason Liu
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Metagenomi Therapeutics Inc
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Metagenomi IP Technologies LLC
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Publication date
Application filed by Metagenomi IP Technologies LLC filed Critical Metagenomi IP Technologies LLC
Priority to CN202080028787.6A priority Critical patent/CN113728098A/zh
Priority to CN202310441977.6A priority patent/CN116515797A/zh
Priority to EP20756259.6A priority patent/EP3924482A4/en
Priority to KR1020217028874A priority patent/KR102623312B1/ko
Priority to CA3130135A priority patent/CA3130135C/en
Priority to AU2020223370A priority patent/AU2020223370B2/en
Priority to JP2021547336A priority patent/JP2022520428A/ja
Priority to KR1020247000311A priority patent/KR20240007322A/ko
Priority to MX2021009886A priority patent/MX2021009886A/es
Priority to US16/917,837 priority patent/US10982200B2/en
Priority to US16/917,838 priority patent/US10913941B2/en
Priority to GB2219441.9A priority patent/GB2617659B/en
Priority to GB2010770.2A priority patent/GB2594339B/en
Publication of WO2020168291A1 publication Critical patent/WO2020168291A1/en
Priority to US17/193,173 priority patent/US12024727B2/en
Priority to US17/402,426 priority patent/US20220220460A1/en
Anticipated expiration legal-status Critical
Priority to US18/335,481 priority patent/US20240117330A1/en
Priority to AU2023206079A priority patent/AU2023206079A1/en
Priority to JP2023146414A priority patent/JP7502537B2/ja
Priority to US18/668,543 priority patent/US20240344045A1/en
Priority to JP2024091759A priority patent/JP2024133476A/ja
Priority to AU2025279720A priority patent/AU2025279720A1/en
Ceased legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
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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 [CRISPR]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • CRISPR Palindromic Repeats
  • RNAs Palindromic Repeats
  • RNAs Palindromic Repeats
  • RNAs Palindromic Repeats
  • RNAs appear to be a pervasive (-45% of bacteria, -84% of archaea) component of prokaryotic immune systems, serving to protect such microorganisms against non-self nucleic acids, such as infectious viruses and plasmids by CRISPR-RNA guided nucleic acid cleavage.
  • DNA deoxyribonucleic acid
  • Cas CRISPR-associated proteins are highly diverse, containing a wide variety of nucleic acid interacting domains.
  • CRISPR DNA elements have been observed as early as 1987, the programmable endonuclease cleavage ability of CRISPR/Cas complexes has only been recognized relatively recently, leading to the use of recombinant CRISPR/Cas systems in diverse DNA manipulation and gene editing applications.
  • the present disclosure provides for an engineered nuclease system, comprising: (a) an endonuclease comprising a RuvC III domain and an HNH domain, wherein the endonuclease is derived from an uncultivated microorganism, wherein the endonuclease is a class 2, type II Cas endonuclease; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.
  • the RuvC III domain comprises a sequence with at least 70%, at least 75%, at least 80% or at least 90% sequence identity to any one of SEQ ID NOs: 1827-3637.
  • an engineered nuclease system comprising: (a) an endonuclease comprising a RuvC III domain having at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.
  • an engineered nuclease system comprising: (a) an endonuclease configured to bind to a protospacer adjacent motif (PAM) sequence comprising SEQ ID NOs: 5512-5537, wherein the endonuclease is a class 2, type II Cas endonuclease; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.
  • PAM protospacer adjacent motif
  • the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease has not been engineered to bind to a different PAM sequence. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Casl4 endonuclease, a Cas 12a endonuclease, a Cas 12b endonuclease, a Cas 12c endonuclease, a Cas 12d endonuclease, a Casl2e endonuclease, a Casl3a endonuclease, a Cas 13b endonuclease, a Casl3c endonuclease, or a Cas 13d endonuclease.
  • the endonuclease has less than 80% identity to a Cas9 endonuclease. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the tracr ribonucleic acid sequence comprises a sequence with at least 80% sequence identity to about 60 to 90 consecutive nucleotides selected from any one of SEQ ID NOs: 5476-5511 and SEQ ID NO: 5538.
  • an engineered nuclease system comprising, (a) an engineered guide ribonucleic acid structure comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to an endonuclease, wherein the tracr ribonucleic acid sequence comprises a sequence with at least 80% sequence identity to about 60 to 90 consecutive nucleotides selected from any one of SEQ ID NOs: 5476-5511 and SEQ ID NO: 5538; and (b) a class 2, type II Cas endonuclease configured to bind to the engineered guide ribonucleic acid.
  • the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence selected from the group comprising SEQ ID NOs: 5512-5537.
  • PAM protospacer adjacent motif
  • the engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides. In some embodiments, the engineered guide ribonucleic acid structure comprises one ribonucleic acid polynucleotide comprising the guide ribonucleic acid sequence and the tracr ribonucleic acid sequence.
  • the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence.
  • the guide ribonucleic acid sequence is 15-24 nucleotides in length.
  • the endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the endonuclease.
  • the NLS comprises a sequence selected from SEQ ID NOs: 5597-5612.
  • the engineered nuclease system further comprises a single- or double-stranded DNA repair template comprising from 5' to 3': a first homology arm comprising a sequence of at least 20 nucleotides 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3' to the target sequence.
  • the first or second homology arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides.
  • the system further comprises a source of Mg 2+
  • the endonuclease and the tracr ribonucleic acid sequence are derived from distinct bacterial species within a same phylum.
  • the endonuclease is derived from a bacterium belonging to a genus Dermabacter.
  • the endonuclease is derived from a bacterium belonging to Phylum
  • the endonuclease is derived from a bacterium comprising a 16S rRNA gene having at least 90% identity to any one of SEQ ID NOs: 5592-5595 .
  • the HNH domain comprises a sequence with at least 70% or at least 80% identity to any one of SEQ ID NOs: 5638-5460. In some embodiments, the
  • endonuclease comprises SEQ ID NOs: 1-1826 or a variant thereof having at least 55% identity thereto.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1827-1830 or SEQ ID NOs: 1827-2140.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 3638-3641 or SEQ ID NOs: 3638-3954. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5615-5632. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-4 or SEQ ID NOs: 1-319.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5461-5464, SEQ ID NOs: 5476-5479, or SEQ ID NOs: 5476-5489.
  • the guide RNA structure comprises an RNA sequence predicted to comprise a hairpin consisting of a stem and a loop, wherein the stem comprises at least 10, at least 12 or at least 14 base-paired
  • the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NOs: 5512-5515 or SEQ ID NOs: 5527- 5530.
  • the endonuclease comprises a sequence at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1827;
  • the guide RNA structure comprises a sequence at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5461 or SEQ ID NO: 5476; and
  • the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5512 or SEQ ID NO: 5527.
  • the endonuclease comprises a sequence at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1828;
  • the guide RNA structure comprises a sequence at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5462 or SEQ ID NO: 5477; and
  • the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5513 or SEQ ID NO: 5528.
  • the endonuclease comprises a sequence at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1829;
  • the guide RNA structure comprises a sequence at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5463 or SEQ ID NO: 5478; and
  • the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5514 or SEQ ID NO: 5529.
  • the endonuclease comprises a sequence at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1830;
  • the guide RNA structure comprises a sequence at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5464 or SEQ ID NO: 5479; and
  • the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5515 or SEQ ID NO: 5530.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 2141-2142 or SEQ ID NOs: 2141-2241. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 3955- 3956 or SEQ ID NOs: 3955-4055. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5632-5638.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 320-321 or SEQ ID NOs: 320-420.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5465, SEQ ID NOs: 5490-5491 or SEQ ID NOs: 5490-5494.
  • the guide RNA structure comprises a tracr ribonucleic acid sequence comprising a hairpin comprising at least 8, at least 10, or at least 12 base-paired ribonucleotides.
  • the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NOs: 5516 and SEQ ID NOs: 5531.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2141;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5490; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5531.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2142;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5465 or SEQ ID NO: 5491; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5516.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 2245-2246. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 4059-4060. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5639-5648.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 424-425.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5498-5499 and SEQ ID NO: 5539.
  • the guide RNA structure comprises a guide ribonucleic acid sequence predicted to comprise a hairpin with an uninterrupted base-paired region comprising at least 8 nucleotides of a guide ribonucleic acid sequence and at least 8 nucleotides of a tracr ribonucleic acid sequence, and wherein the tracr ribonucleic acid sequence comprises, from 5’ to 3’, a first hairpin and a second hairpin, wherein the first hairpin has a longer stem than the second hairpin.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 2242-2244 or SEQ ID NOs: 2247-2249. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 4056- 4058 and SEQ ID NOs 4061-4063. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5639-5648.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 421-423 or SEQ ID NOs: 426-428.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5466-5467, SEQ ID NOs: 5495-5497, SEQ ID NO: 5500-5502, and SEQ ID NO: 5539.
  • the guide RNA structure comprises a guide ribonucleic acid sequence predicted to comprise a hairpin with an uninterrupted base-paired region comprising at least 8 nucleotides of a guide ribonucleic acid sequence and at least 8 nucleotides of a tracr ribonucleic acid sequence, and wherein the tracr ribonucleic acid sequence comprises, from 5’ to 3’, a first hairpin and a second hairpin, wherein the first hairpin has a longer stem than the second hairpin.
  • the endonuclease is configured to binding to a PAM comprising a sequence selected from the group consisting of SEQ ID NOs: 5517-5518 or SEQ ID NOs: 5532-5534. In some embodiments: (a) the endonuclease comprises a sequence at least 70%,
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5500; and (c) the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5517 or SEQ ID NO: 5532.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2248;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5501; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5518 or SEQ ID NOs: 5533.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2248;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5501;
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5518 or SEQ ID NOs: 5533.
  • endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2249;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5502; and (c) the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5534.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2253 or SEQ ID NOs: 2253-2481. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4067 or SEQ ID NOs: 4067-4295. In some embodiments, the endonuclease comprises a peptide motif according to SEQ ID NO: 5649. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO:
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5468 or SEQ ID NO: 5503.
  • the endonuclease is configured to binding to a PAM comprising a sequence selected from the group consisting of SEQ ID NOs: 5519.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2253;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5468 or SEQ ID NO: 5503; and (c) the
  • endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5519.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 2482-2489. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 4296-4303. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of or SEQ ID NOs: 661-668. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of or SEQ ID NOs: 2490-2498. In some embodiments, the
  • endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 4304-4312. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 669-677. In some embodiments, the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5504.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2499 or SEQ ID NOs: 2499-2750. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4313 or SEQ ID NOs: 4313-4564. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5650-5667.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 678 or SEQ ID NOs: 678-929.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5469 or SEQ ID NO: 5505.
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NOs: 5520 or SEQ ID NOs: 5535.
  • (a) the endonuclease comprises a sequence at least 70%,
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5469 or SEQ ID NO: 5505; and (c) the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5520 or SEQ ID NO: 5535.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2751 or SEQ ID NOs: 2751-2913. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4565 or SEQ ID NOs: 4565-4727. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs:
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 930 or SEQ ID NOs: 930-1092.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5470 or SEQ ID NOs: 5506.
  • the endonuclease is configured to binding to a PAM comprising a sequence selected from the group consisting of SEQ ID NOs: 5521 or SEQ ID NOs: 5536.
  • (a) the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2751;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5470 or SEQ ID NO: 5506; and (c) the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5521 or SEQ ID NO: 5536.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2914 or SEQ ID NOs: 2914-3174. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4728 or SEQ ID NOs: 4728-4988. In some embodiments, the endonuclease comprises at least 1, at least 2, or at least 3 peptide motifs selected from the group consisting of SEQ ID NOs: 5676-5678. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1093 or SEQ ID NOs: 1093-1353.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5471, SEQ ID NO: 5507, and SEQ ID NOs: 5540-5542.
  • the guide RNA structure comprises a tracr ribonucleic acid sequence predicted to comprise at least two hairpins comprising less than 5 base-paired ribonucleotides.
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5522.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2914;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5471 or SEQ ID NO: 5507; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5522.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3175 or SEQ ID NOs: 3175-3330. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4989 or SEQ ID NOs: 4989-5146. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5679-5686.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1354 or SEQ ID NOs: 1354-1511.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5472 or SEQ ID NOs: 5508.
  • the endonuclease is configured to binding to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5523 or SEQ ID NO: 5537.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3175;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5472 or SEQ ID NO: 5508; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5523 or SEQ ID NO: 5537.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 3331 or SEQ ID NOs: 3331-3474. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5147 or SEQ ID NOs: 5147-5290. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5674-5675 and SEQ ID NOs: 5687-5693.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1512 or SEQ ID NOs: 1512-1655.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5473 or SEQ ID NO: 5509.
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5524.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3331;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5473 or SEQ ID NO: 5509; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5524.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3475 or SEQ ID NOs: 3475-3568.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5291 or SEQ ID NOs: 5291-5389. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs:
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1656 or SEQ ID NOs: 1656-1755.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5474 or SEQ ID NO: 5510.
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NOs: 5525.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3475;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5474 or SEQ ID NO: 5510; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5525.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3569 or SEQ ID NOs: 3569-3637. In some embodiments, the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5390 or SEQ ID NOs: 5390-5460. In some embodiments, the endonuclease comprises at least 1, at least 2, at least 3, at least 4, or at least 5 peptide motifs selected from the group consisting of SEQ ID NOs: 5700-5717.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1756 or SEQ ID NOs: 1756-1826.
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5475 or SEQ ID NOs: 5511.
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5526.
  • the endonuclease comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3569;
  • the guide RNA structure comprises a sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5475 or SEQ ID NO: 5511; and
  • the endonuclease is configured to binding to a PAM comprising SEQ ID NO: 5526.
  • the sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT, or Smith -Waterman homology search algorithm.
  • sequence identity is determined by the BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • an engineered guide ribonucleic acid polynucleotide comprising: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule; and (b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double- stranded RNA (dsRNA) duplex, wherein the two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides, and wherein the engineered guide ribonucleic acid polynucleotide is configured to forming a complex with an endonuclease comprising a RuvC III domain having at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637 and targeting the complex to the target sequence of the target DNA molecule.
  • dsRNA double- stranded RNA
  • the DNA-targeting segment is positioned 5' of both of the two
  • the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5476-5479 or SEQ ID NOs: 5476-5489;
  • the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of (SEQ ID NOs: 5490-5491 or SEQ ID NOs: 5490-5494) and SEQ ID NO: 5538;
  • the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5498-5499;
  • the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5495-5497 and SEQ ID NOs: 5500-5502;
  • the protein binding segment comprises a sequence having at least 70%, at least 80%
  • the guide ribonucleic acid polynucleotide comprises an RNA sequence comprising a hairpin comprising a stem and a loop, wherein the stem comprises at least 10, at least 12, or at least 14 base-paired ribonucleotides, and an asymmetric bulge within 4 base pairs of the loop;
  • the guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence predicted to comprise a hairpin comprising at least 8, at least 10, or at least 12 base- paired ribonucleotides;
  • the guide ribonucleic acid polynucleotide comprises a guide ribonucleic acid sequence predicted to comprise a hairpin with an uninterrupted base-paired region comprising at least 8 nucleotides of a guide ribonucleic acid sequence and at least 8 nucleotides of a tracr ribonucleic acid sequence, and wherein the tracr
  • the present disclosure provides for a deoxyribonucleic acid
  • polynucleotide encoding any of the engineered guide ribonucleic acid polynucleotides described herein.
  • the present disclosure provides for a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein the nucleic acid encodes a class 2, type II Cas endonuclease comprising a RuvC III domain and an HNH domain, and wherein the endonuclease is derived from an uncultivated microorganism.
  • the present disclosure provides for a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein the nucleic acid encodes an endonuclease comprising a RuvC III domain having at least 70% sequence identity to any one of SEQ ID NOs: 1827-3637.
  • the endonuclease comprises an HNH domain having at least 70% or at least 80% sequence identity to any one of SEQ ID NOs: 3638-5460.
  • the endonuclease comprises SEQ ID NOs: 5572-5591 or a variant thereof having at least 70% sequence identity thereto.
  • the endonuclease comprises a sequence encoding one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the endonuclease.
  • NLS nuclear localization sequences
  • the NLS comprises a sequence selected from SEQ ID NOs: 5597-5612.
  • the organism is prokaryotic, bacterial, eukaryotic, fungal, plant, mammalian, rodent, or human.
  • the organism is E. coli , and: (a) the nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5572-5575; (b) the nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5576- 5577; (c) the nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5578-5580; (d) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5581; (e) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5582; (f) the nucleic acid sequence has
  • the organism is human, and: (a) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5588 or SEQ ID NO: 5589; or (b) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5590 or SEQ ID NO: 5591.
  • the present disclosure provides for a vector comprising a nucleic acid sequence encoding a class 2, type II Cas endonuclease comprising a RuvC III domain and an HNH domain, wherein the endonuclease is derived from an uncultivated microorganism.
  • the present disclosure provides for a vector comprising the any of the nucleic acids described herein.
  • the vector further comprises a nucleic acid encoding an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease comprising: (a) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (b) a tracr ribonucleic acid sequence configured to binding to the endonuclease.
  • the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV) derived virion, or a lentivirus.
  • AAV adeno-associated virus
  • the present disclosure provides for a cell comprising any of the vectors described herein.
  • the present disclosure provides for a method of manufacturing an endonuclease, comprising cultivating any of the cells described herein.
  • the present disclosure provides for a method for binding, cleaving, marking, or modifying a double-stranded deoxyribonucleic acid polynucleotide, comprising: (a) contacting the double-stranded deoxyribonucleic acid polynucleotide with a class 2, type II Cas endonuclease in complex with an engineered guide ribonucleic acid structure configured to bind to the endonuclease and the double-stranded deoxyribonucleic acid polynucleotide; (b) wherein the double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer adjacent motif (PAM); and (c) wherein the PAM comprises a sequence selected from the group consisting of SEQ ID NOs: 5512-5526 or SEQ ID NOs: 5527-5537.
  • PAM protospacer adjacent motif
  • the double- stranded deoxyribonucleic acid polynucleotide comprises a first strand comprising a sequence complementary to a sequence of the engineered guide ribonucleic acid structure and a second strand comprising the PAM.
  • the PAM is directly adjacent to the 3' end of the sequence complementary to the sequence of the engineered guide ribonucleic acid structure.
  • the class 2, type II Cas endonuclease is not a Cas9 endonuclease, a Casl4 endonuclease, a Cas 12a endonuclease, a Cas 12b endonuclease, a Cas 12c endonuclease, a Cas 12d endonuclease, a Casl2e endonuclease, a Casl3a endonuclease, a Cas 13b endonuclease, a Casl3c endonuclease, or a Cas 13d endonuclease.
  • the class 2, type II Cas endonuclease is derived from an uncultivated microorganism.
  • the double-stranded deoxyribonucleic acid polynucleotide is a eukaryotic, plant, fungal, mammalian, rodent, or human double-stranded deoxyribonucleic acid polynucleotide.
  • the PAM comprises a sequence selected from the group consisting of SEQ ID NOs: 5512-5515 and SEQ ID NOs: 5527-5530;
  • the PAM comprises SEQ ID NO: 5516 or SEQ ID NO: 5531;
  • the PAM comprises SEQ ID NO: 5539;
  • the PAM comprises SEQ ID NO: 5517 or SEQ ID NO: 5518;
  • the PAM comprises SEQ ID NO: 5519;
  • the PAM comprises SEQ ID NO: 5520 or SEQ ID NO: 5535;
  • the PAM comprises SEQ ID NO: 5521 or SEQ ID NO: 5536;
  • the PAM comprises SEQ ID NO: 5522;
  • the PAM comprises SEQ ID NO: 5523 or SEQ ID NO: 5537;
  • the PAM comprises SEQ ID NO: 5524;
  • the PAM comprises SEQ ID NO: 5525; or (1) the PAM comprises SEQ ID NO: 5526.
  • the present disclosure provides for a method of modifying a target nucleic acid locus, the method comprising delivering to the target nucleic acid locus any of the engineered nuclease systems described herein, wherein the endonuclease is configured to form a complex with the engineered guide ribonucleic acid structure, and wherein the complex is configured such that upon binding of the complex to the target nucleic acid locus, the complex modifies the target nucleic locus.
  • modifying the target nucleic acid locus comprises binding, nicking, cleaving, or marking the target nucleic acid locus.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus is in vitro. In some embodiments, the target nucleic acid locus is within a cell.
  • the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell.
  • delivering the engineered nuclease system to the target nucleic acid locus comprises delivering the nucleic acid of any of claims 135-140 or the vector of any of claims 142-146. In some embodiments, delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a nucleic acid comprising an open reading frame encoding the endonuclease. In some embodiments, the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the engineered nuclease system to the target nucleic acid locus comprises delivering a capped mRNA containing the open reading frame encoding the endonuclease. In some embodiments, the engineered nuclease system to the target nucleic acid locus comprises delivering a translated polypeptide. In some embodiments, the engineered nuclease system to the target nucleic acid locus comprises delivering a deoxyribonucleic acid (DNA) encoding the engineered guide ribonucleic acid structure operably linked to a ribonucleic acid (RNA) pol III promoter. In some embodiments, the endonuclease induces a single-stranded break or a double- stranded break at or proximal to the target locus.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the endonuclease induces a single-stranded break or a double- stranded break at or
  • FIGURE 1 depicts typical organizations of CRISPR/Cas loci of different classes and types.
  • FIGURE 2 depicts the architecture of a natural Class2/Type II crRNA/tracrRNA pair, compared to a hybrid sgRNA wherein both are joined.
  • FIGURE 3 depicts schematics showing organization of CRISPR loci encoding enzymes from the MG1 family.
  • FIGURE 4 depicts schematics showing organization of CRISPR loci encoding enzymes from the MG2 family.
  • FIGURE 5 depicts schematics showing organization of CRISPR loci encoding enzymes from the MG3 family.
  • FIGURE 6 depicts a structure-based alignment of an enzyme of the present disclosure (MGl-1) versus Cas9 from Staphylococcus aureus (SEQ ID NO:5613).
  • FIGURE 7 depicts a structure-based alignment of an enzyme of the present disclosure (MG2-1) versus Cas9 from Staphylococcus aureus (SEQ ID NO:5613).
  • FIGURE 8 depicts a structure-based alignment of an enzyme of the present disclosure (MG3-1) versus Cas9 from Actinomyces naeslundii (SEQ ID NO: 5614).
  • FIGURES 9A, 9B, 9C, 9D, 9E, 9F, 9G, and 9H depicts a structure-based alignment of MG1 family enzymes MGl-1 through MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3).
  • FIGURE 10 depicts in vitro cleavage of DNA by MG1-4 in complex with its
  • FIGURE 11 depicts in cell cleavage of E. coli genomic DNA using MG1-4 along with its corresponding sgRNA. Shown are dilution series of cells transformed with MG1-4 along with target or non-target spacer (top); bottom panel shows the data quantitated, where the left bar represents non-target sgRNA and the right bar represents target sgRNA.
  • FIGURE 12 depicts in cell indel formation generated by transfection of HEK cells with MG1-4 or MG1-6 constructs described in Example 11 alongside their corresponding sgRNAs containing various different targeting sequences targeting various locations in the human genome.
  • FIGURE 13 depicts vitro cleavage of DNA by MG3-6 in complex with its corresponding sgRNA containing targeting sequences of varying lengths.
  • FIGURE 14 depicts in cell cleavage of E. coli genomic DNA using MG3-7 along with its corresponding sgRNA. Shown are dilution series of cells transformed with MG3-7 along with target or non-target spacer (top); bottom panel shows the data quantitated, where the left bar represents non-target sgRNA and the right bar represents target sgRNA.
  • FIGURE 15 depicts in cell indel formation generated by transfection of HEK cells with MG3-7 constructs described in Example 13 alongside their corresponding sgRNAs containing various different targeting sequences targeting various locations in the human genome.
  • FIGURE 16 depicts in vitro cleavage of DNA by MG15-1 in complex with its corresponding sgRNA containing targeting sequences of varying lengths.
  • FIGURES 17, 18, 19, and 20 depict agarose gels showing the results of PAM vector library cleavage in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNAs or sgRNAs.
  • FIGURES 21, 22, 23, 24, 25 and 26 depict predicted structures (predicted e.g., as in Example 7) of corresponding sgRNAs of MG enzymes described herein.
  • FIGURES 27, 28, 29, 30, 31, 32 and 33 depict seqLogo representations of PAM sequences derived via NGS as described herein (e.g. as described in Example 6).
  • FIGURE 34 depicts in cell cleavage of E. coli genomic DNA using MG2-7 along with its corresponding sgRNA. Shown are dilution series of cells transformed with MG2-7 along with target or non-target spacer (top); bottom panel shows the data quantitated, where the right bar represents non-target sgRNA and the left bar represents target sgRNA.
  • FIGURE 35 depicts in cell cleavage of E. coli genomic DNA using MG14-1 along with its corresponding sgRNA. Shown are dilution series of cells transformed with MG14-1 along with target or non-target spacer (top); bottom panel shows the data quantitated, where the right bar represents non-target sgRNA and the left bar represents target sgRNA.
  • FIGURE 36 depicts in cell cleavage of E. coli genomic DNA using MG15-1 along with its corresponding sgRNA. Shown are dilution series of cells transformed with MG15-1 along with target or non-target spacer (top); bottom panel shows the data quantitated, where the right bar represents non-target sgRNA and the left bar represents target sgRNA.
  • SEQ ID NOs: 1-319 show the full-length peptide sequences of MG1 nucleases.
  • SEQ ID NOs: 1827-2140 show the peptide sequences of RuvC III domains of MG1 nucleases above.
  • SEQ ID NOs: 3638-3955 show the peptide of HNH domains of MG1 nucleases above.
  • SEQ ID NOs: 5476-5479 show the nucleotide sequences of MG1 tracrRNAs derived from the same loci as MG1 nucleases above (e.g., same loci as SEQ ID NO: 1-4, respectively).
  • SEQ ID NOs: 5461-5464 show the nucleotide sequences of sgRNAs engineered to function with an MG1 nuclease (e.g., SEQ ID NO: 1-4, respectively), where Ns denote nucleotides of a targeting sequence.
  • SEQ ID NOs: 5572-5575 show nucleotide sequences for A. coli codon-optimized coding sequences for MG1 family enzymes (SEQ ID NOs: 1-4).
  • SEQ ID NOs: 5588-5589 show nucleotide sequences for human codon-optimized coding sequences for MG1 family enzymes (SEQ ID NOs: 1 and 3).
  • SEQ ID NOs: 5616-5632 show peptide motifs characteristic of MG1 family enzymes.
  • SEQ ID NOs: 320-420 show the full-length peptide sequences of MG2 nucleases.
  • SEQ ID NOs: 2141-2241 show the peptide sequences of RuvC III domains of MG2 nucleases above.
  • SEQ ID NOs: 3955-4055 show the peptide of HNH domains of MG2 nucleases above.
  • SEQ ID NOs: 5490-5494 show the nucleotide sequences of MG2 tracrRNAs derived from the same loci as MG2 nucleases above (e.g., same loci as SEQ ID NOs: 320, 321, 323, 325, and 326, respectively).
  • SEQ ID NO: 5465 shows the nucleotide sequence of an sgRNA engineered to function with an MG2 nuclease (e.g., SEQ ID NO: 321 above).
  • SEQ ID NOs: 5572-5575 show nucleotide sequences for A. coli codon-optimized coding sequences for MG2 family enzymes.
  • SEQ ID NOs: 5631-5638 show peptide sequences characteristic of MG2 family enzymes.
  • SEQ ID NOs: 421-431 show the full-length peptide sequences of MG3 nucleases.
  • SEQ ID NOs: 2242-2251 show the peptide sequences of RuvC III domains of MG3 nucleases above.
  • SEQ ID NOs: 4056-4066 show the peptide of HNH domains of MG3 nucleases above.
  • SEQ ID NOs: 5495-5502 show the nucleotide sequences of MG3 tracrRNAs derived from the same loci as MG3 nucleases above (e.g., same loci as SEQ ID NOs: 421-428, respectively).
  • SEQ ID NOs: 5466-5467 show the nucleotide sequence of sgRNAs engineered to function with an MG3 nuclease (e.g., SEQ ID NOs: 421 - 423).
  • SEQ ID NOs: 5578-5580 show nucleotide sequences for A. coli codon-optimized coding sequences for MG3 family enzymes.
  • SEQ ID NOs: 5639-5648 show peptide sequences characteristic of MG3 family enzymes.
  • SEQ ID NOs: 432-660 show the full-length peptide sequences of MG4 nucleases.
  • SEQ ID NOs: 2253-2481 show the peptide sequences of RuvC III domains of MG4 nucleases above.
  • SEQ ID NOs: 4067-4295 show the peptide of HNH domains of MG4 nucleases above.
  • SEQ ID NO: 5503 shows the nucleotide sequences of an MG4 tracrRNA derived from the same loci as MG4 nucleases above.
  • SEQ ID NO: 5468 shows the nucleotide sequence of sgRNAs engineered to function with an MG4 nuclease.
  • SEQ ID NO: 5649 shows a peptide sequence characteristic of MG4 family enzymes.
  • SEQ ID NOs: 661-668 show the full-length peptide sequences of MG6 nucleases.
  • SEQ ID NOs: 2482-2489 show the peptide sequences of RuvC III domains of MG6 nucleases above.
  • SEQ ID NOs: 4296-4303 show the peptide of HNH domains of MG3 nucleases above.
  • SEQ ID NOs: 669-677 show the full-length peptide sequences of MG7 nucleases.
  • SEQ ID NOs: 2490-2498 show the peptide sequences of RuvC III domains of MG7 nucleases above.
  • SEQ ID NOs: 4304-4312 show the peptide of HNH domains of MG3 nucleases above.
  • SEQ ID NO: 5504 shows the nucleotide sequence of an MG7 tracrRNA derived from the same loci as MG7 nucleases above.
  • SEQ ID NOs: 678-929 show the full-length peptide sequences of MG14 nucleases.
  • SEQ ID NOs: 2499-2750 show the peptide sequences of RuvC III domains of MG14 nucleases above.
  • SEQ ID NOs: 4313-4564 show the peptide of HNH domains of MG14 nucleases above.
  • SEQ ID NO: 5505 shows the nucleotide sequences of MG14 tracrRNA derived from the same loci as MG14 nucleases above.
  • SEQ ID NO: 5581 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG14 family enzyme.
  • SEQ ID NOs: 5650-5667 show peptide sequences characteristic of MG14 family enzymes.
  • SEQ ID NOs: 930-1092 show the full-length peptide sequences of MG15 nucleases.
  • SEQ ID NOs: 2751-2913 show the peptide sequences of RuvC III domains of MG15 nucleases above.
  • SEQ ID NOs: 4565-4727 show the peptide of HNH domains of MG15 nucleases above.
  • SEQ ID NO: 5506 shows the nucleotide sequences of MG15 tracrRNA derived from the same loci as MG15 nucleases above.
  • SEQ ID NOs: 5470 shows the nucleotide sequence of an sgRNA engineered to function with an MG15 nuclease.
  • SEQ ID NO: 5582 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG15 family enzyme.
  • SEQ ID NOs: 5668-5675 show peptide sequences characteristic of MG15 family enzymes.
  • SEQ ID NOs: 1093-1353 show the full-length peptide sequences of MG16 nucleases.
  • SEQ ID NOs: 2914-3174 show the peptide sequences of RuvC III domains of MG16 nucleases above.
  • SEQ ID NOs: 4728-4988 show the peptide of HNH domains of MG16 nucleases above.
  • SEQ ID NOs: 5507 show the nucleotide sequences of an MG16 tracrRNA derived from the same loci as MG3 nucleases above.
  • SEQ ID NOs: 5471 shows the nucleotide sequence of sgRNAs engineered to function with an MG16 nuclease.
  • SEQ ID NO: 5583 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG16 family enzyme.
  • SEQ ID NOs: 5676-5678 show peptide sequences characteristic of MG16 family enzymes.
  • SEQ ID NOs: 1354-1511 show the full-length peptide sequences of MG18 nucleases.
  • SEQ ID NOs: 3175-3330 show the peptide sequences of RuvC III domains of MG18 nucleases above.
  • SEQ ID NOs: 4989-5146 show the peptide of HNH domains of MG18 nucleases above.
  • SEQ ID NO: 5508 shows the nucleotide sequences of MG18 tracrRNA derived from the same loci as MG18 nucleases above.
  • SEQ ID NOs: 5472 shows the nucleotide sequence of an sgRNA engineered to function with an MG18 nuclease.
  • SEQ ID NO: 5584 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG18 family enzyme.
  • SEQ ID NOs: 5679-5686 show peptide sequences characteristic of MG18 family enzymes.
  • SEQ ID NOs: 1512-1655 show the full-length peptide sequences of MG21 nucleases.
  • SEQ ID NOs: 3331-3474 show the peptide sequences of RuvC III domains of MG21 nucleases above.
  • SEQ ID NOs: 5147-5290 show the peptide of HNH domains of MG21 nucleases above.
  • SEQ ID NOs: 5509 show the nucleotide sequence of an MG21 tracrRNA derived from the same loci as MG21 nucleases above.
  • SEQ ID NOs: 5473 shows the nucleotide sequence of an sgRNA engineered to function with an MG21 nuclease.
  • SEQ ID NO: 5585 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG21 family enzyme.
  • SEQ ID NOs: 5687-5692 and 5674-5675 show peptide sequences characteristic of MG21 family enzymes.
  • SEQ ID NOs: 1656-1755 show the full-length peptide sequences of MG22 nucleases.
  • SEQ ID NOs: 3475-3568 show the peptide sequences of RuvC III domains of MG22 nucleases above.
  • SEQ ID NOs: 5291-5389 show the peptide of HNH domains of MG22 nucleases above.
  • SEQ ID NO: 5510 show the nucleotide sequence of an MG22 tracrRNA derived from the same loci as MG22 nucleases above.
  • SEQ ID NOs: 5474 shows the nucleotide sequence of an sgRNAs engineered to function with an MG22 nuclease.
  • SEQ ID NO: 5586 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG22 family enzyme.
  • SEQ ID NOs: 5694-5699 show peptide sequences characteristic of MG22 family enzymes.
  • SEQ ID NOs: 1756-1826 show the full-length peptide sequences of MG23 nucleases.
  • SEQ ID NOs: 3569-3637 show the peptide sequences of RuvC III domains of MG23 nucleases above.
  • SEQ ID NOs: 5390-5460 show the peptide of HNH domains of MG23 nucleases above.
  • SEQ ID NO: 5511 shows the nucleotide sequences of an MG23 tracrRNA derived from the same loci as MG23 nucleases above.
  • SEQ ID NOs: 5475 shows the nucleotide sequence of an sgRNA engineered to function with an MG23 nuclease.
  • SEQ ID NO: 5587 shows a nucleotide sequence for an E. coli codon-optimized coding sequences for an MG23 family enzyme.
  • SEQ ID NOs: 5700-5717 show peptide sequences characteristic of MG23 family enzymes. DETAILED DESCRIPTION
  • “about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within one or more than one standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.
  • a“cell” generally refers to a biological cell.
  • a cell may be the basic structural, functional and/or biological unit of a living organism.
  • a cell may originate from any organism having one or more cells.
  • Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, homworts, liverworts, mosses), an algal cell, (e.g. context Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gadit
  • seaweeds e.g., kelp
  • a fungal cell e.g.,, a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide may comprise a synthetic nucleotide.
  • a nucleotide may comprise a synthetic nucleotide analog.
  • Nucleotides may be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • the term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside
  • triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives may include, for example, [aSJdATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores). Labeling may also be carried out with quantum dots.
  • Detectable labels may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6- carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS).
  • fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP,
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically-modified single nucleotide can be biotin-dNTP.
  • biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6- ddATP, biotin- 14-d ATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin- 11-dUTP, biotin- 16-dUTP, biotin-20-dUTP).
  • a polynucleotide may be exogenous or endogenous to a cell.
  • a polynucleotide may exist in a cell-free environment.
  • a polynucleotide may be a gene or fragment thereof.
  • a polynucleotide may be DNA.
  • a polynucleotide may be RNA.
  • a polynucleotide may have any three-dimensional structure and may perform any function.
  • a polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids,
  • dideoxynucleotides cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • fluorophores e.g., rhodamine or fluorescein linked to the sugar
  • thiol containing nucleotides biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • the terms“transfection” or“transfected” generally refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods.
  • the nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.
  • the terms“peptide,”“polypeptide,” and“protein” are used interchangeably herein to generally refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains).
  • amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and“amino acids,” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues.
  • Modified amino acids may include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • Amino acid analogues may refer to amino acid derivatives.
  • amino acid derivatives includes both D-amino acids and L-amino acids.
  • non-native can generally refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein.
  • Non-native may refer to affinity tags.
  • Non-native may refer to fusions.
  • Non-native may refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions.
  • a non-native sequence may exhibit and/or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) that may also be exhibited by the nucleic acid and/or polypeptide sequence to which the non-native sequence is fused.
  • a non-native nucleic acid or polypeptide sequence may be linked to a naturally- occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and/or polypeptide sequence encoding a chimeric nucleic acid and/or polypeptide.
  • promoter generally refers to the regulatory DNA region which controls transcription or expression of a gene and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated.
  • a promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription.
  • A‘basal promoter’ also referred to as a‘core promoter’, may generally refer to a promoter that contains all the basic necessary elements to promote transcriptional expression of an operably linked polynucleotide.
  • Eukaryotic basal promoters typically, though not necessarily, contain a TATA-box and/or a CAAT box.
  • the term“expression”, as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • “operably linked”,“operable linkage”,“operatively linked”, or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner.
  • a regulatory element which may comprise promoter and/or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
  • A“vector” as used herein generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell.
  • vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • the vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.
  • an expression cassette and“a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression.
  • an expression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.
  • A“functional fragment” of a DNA or protein sequence generally refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence.
  • a biological activity of a DNA sequence may be its ability to influence expression in a manner known to be attributed to the full- length sequence.
  • an“engineered” object generally indicates that the object has been modified by human intervention.
  • a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property.
  • An“engineered” system comprises at least one engineered component.
  • “synthetic” and“artificial” are used interchangeably to refer to a protein or a domain thereof that has low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein.
  • VPR and VP64 domains are synthetic transactivation domains.
  • tracrRNA or“tracr sequence”, as used herein, can generally refer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and/or sequence similarity to a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S. pyogenes S. aureus, etc or SEQ ID NOs: 5476-5511).
  • tracrRNA can refer to a nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
  • tracrRNA may refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera.
  • a tracrRNA may refer to a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.
  • a tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.
  • Type II tracrRNA sequences can be predicted on a genome sequence by identifying regions with complementarity to part of the repeat sequence in an adjacent CRISPR array.
  • a“guide nucleic acid” can generally refer to a nucleic acid that may hybridize to another nucleic acid.
  • a guide nucleic acid may be RNA.
  • a guide nucleic acid may be DNA.
  • the guide nucleic acid may be programmed to bind to a sequence of nucleic acid site- specifically.
  • the nucleic acid to be targeted, or the target nucleic acid may comprise nucleotides.
  • the guide nucleic acid may comprise nucleotides.
  • a portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid.
  • the strand of a double- stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid may be called the complementary strand.
  • a guide nucleic acid may comprise a polynucleotide chain and can be called a“single guide nucleic acid.”
  • a guide nucleic acid may comprise two polynucleotide chains and may be called a“double guide nucleic acid.” If not otherwise specified, the term“guide nucleic acid” may be inclusive, referring to both single guide nucleic acids and double guide nucleic acids.
  • a guide nucleic acid may comprise a segment that can be referred to as a“nucleic acid-targeting segment” or a“nucleic acid-targeting sequence.”
  • a nucleic acid-targeting segment may comprise a sub-segment that may be referred to as a“protein binding segment” or“protein binding sequence” or“Cas protein binding segment”.
  • sequence identity in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm.
  • Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https://blast.ncbi.nlm.nih.gov); CLUSTALW with parameters of ; the Smith-Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAFFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign
  • RuvC III domain generally refers to a third discontinuous segment of a RuvC endonuclease domain (the RuvC nuclease domain being comprised of three discontiguous segments, RuvC I, RuvC II, and RuvC III).
  • a RuvC domain or segments thereof can generally be identified by alignment to known domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on known domain sequences (e.g., Pfam HMM PF18541 for RuvC III).
  • HNH domain generally refers to an endonuclease domain having characteristic histidine and asparagine residues.
  • An HNH domain can generally be identified by alignment to known domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on known domain sequences (e.g., Pfam HMM PF01844 for domain HNH).
  • HMMs Hidden Markov Models
  • CRISPR/Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes.
  • CRISPR/Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30-40bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory proteins/enzymes.
  • Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome).
  • PAM protospacer-adjacent motif
  • CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.
  • Type I CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.
  • Type I CRISPR-Cas systems are considered of moderate complexity in terms of components.
  • pre-crRNA long precursor crRNA
  • PAM protospacer-adjacent motif
  • Cas6 endoribonuclease subunit
  • Cascade a large endonuclease complex
  • Cas3 a nuclease protein component of the crRNA- directed nuclease complex.
  • Cas I nucleases function primarily as DNA nucleases.
  • Type III CRISPR systems may be characterized by the presence of a central nuclease, known as CaslO, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits.
  • CaslO central nuclease
  • RAMP repeat-associated mysterious protein
  • the mature crRNA is processed from a pre- crRNA using a Cas6-like enzyme.
  • type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).
  • Type IV CRISPR-Cas systems possess an effector complex that consists of a highly reduced large subunit nuclease (csfl), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.
  • csfl highly reduced large subunit nuclease
  • csf3 two genes for RAMP proteins of the Cas5
  • csf2 Cas7
  • Class II CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.
  • Type II CRISPR-Cas systems are considered the simplest in terms of components.
  • the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g. Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA.
  • Cas II nucleases are known as DNA nucleases.
  • Type 2 effectors generally exhibit a structure consisting of a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain.
  • the RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.
  • Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g. Casl2) structure similar to that of Type II effectors, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again known as DNA nucleases.
  • Casl2 nuclease effector
  • Type V enzymes e.g., Casl2a
  • Casl2a some Type V enzymes appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.
  • Type VI CRIPSR-Cas systems have RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (e.g. Casl3) comprises two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to not need a tracrRNA for processing of pre-crRNA into crRNA. Similar to type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.
  • C2C2C2C2C2C2 some Type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.
  • Class II CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease/genome editing applications.
  • Jinek et al. Science. 2012 Aug 17;337(6096):816-21, which is entirely incorporated herein by reference.
  • the Jinek study first described a system that involved (i) recombinantly-expressed, purified full- length Cas9 (e.g., a Class II, Type II Cas enzyme) isolated from S.
  • Cas9 e.g., a Class II, Type II Cas enzyme
  • pyogenes SF370 (ii) purified mature ⁇ 42 nt crRNA bearing a ⁇ 20 nt 5’ sequence complementary to the target DNA sequence desired to be cleaved followed by a 3’ tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg2+.
  • a linker e.g., GAAA
  • sgRNA single fused synthetic guide RNA
  • MG1 Enzymes are complementary targeting nucleic acid sequence joined to a 3’ tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g., the U6 promoter) .
  • a suitable Polymerase III promoter e.g., the U6 promoter
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 1827-2140.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 1827-2140.
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 1827-2140.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 1827-1831.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 1827-1831.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 1827-1831.
  • the endonuclease may comprise a RuvC III domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to SEQ ID NO: 1827.
  • the endonuclease may comprise a RuvC III domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to SEQ ID NO: 1828.
  • the endonuclease may comprise a RuvC III domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to SEQ ID NO: 1829.
  • the endonuclease may comprise a RuvC III domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to SEQ ID NO: 1830.
  • the endonuclease may comprise a RuvC III domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to SEQ ID NO: 1831.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638-3955.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3638-3955.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3638-3955.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638- 3955.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3638-3955.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3638-3955.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638-3641.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3638-3641.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3638-3641.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3638.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3638.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3639.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3639.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3639.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3640.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3640.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3640.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3641.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3641.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3641.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-6 or 9-319 .
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1-6 or 9- 319.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-4.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1-4.
  • the endonuclease may comprise a peptide motif substantially identical to any one of SEQ ID NOs: 5615, 5616, or 5617.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 1-6 or 9-319, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-319.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 below, or a combination thereof:
  • Table 1 Example NLS Sequences that can be used with Cas Effectors According to the Disclosure
  • the endonuclease may be recombinant (e.g., cloned, expressed, and purified by a suitable method such as expression in E. cob followed by epitope-tag purification).
  • the endonuclease may be derived from a bacterium with a 16S rRNA gene having at least about 90% identity to any one of SEQ ID NOs: 5592-5595.
  • the endonuclease may be derived from a species having a 16S rRNA gene at least about 80%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 5592-5595.
  • the endonuclease may be derived from a species having a 16S rRNA gene substantially identical to any one of SEQ ID NOs: 5592-5595.
  • the endonuclease may be derived from a bacterium belonging to the Phylum Verrucomicrobia or the Phylum Candidatus Peregrinibacteria.
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5476-5489.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5476-5489.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5476-5489.
  • the tracrRNA may comprise any of SEQ ID NOs: 5476-5489.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to any one of SEQ ID NOs: 5461-5464.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 5461-5464.
  • the sgRNA may comprise a sequence substantially identical to any one of SEQ ID NOs: 5461-5464.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus, may modify the target nucleic acid locus.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double- stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 1827- 2140.
  • RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 1827- 2140.
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to any of SEQ ID NOs: 5572-5575 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure may provide for an expression cassette comprising the system disclosed herein, or the nucleic acid described herein.
  • the expression cassette or nucleic acid may be supplied as a vector.
  • the expression cassette, nucleic acid, or vector may be supplied in a cell.
  • the cell is a cell of a bacterium with a 16S rRNA gene having at least about 90% (e.g., at least about 99%) identity to any one of SEQ ID NOs: 5592-5595.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2141-2241.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2141-2241.
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2141-2142.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2141-2142.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2141-2142.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2141-2142.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3955-4055.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3955-4055.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3955-4055.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3955- 3956.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 3955-3956.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 3955-3956.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 320-420 .
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 320-420.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs:320-321.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 320-321.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 320-420, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 320-420.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5490-5494.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5490-5494.
  • 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5490-5494.
  • the tracrRNA may comprise any of SEQ ID NOs: 5490-5494.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5465.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5465.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5465.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’ : a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lk
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2141- 2241.
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to any of SEQ ID NOs: 5576-5577 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2242-2251.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2242-2251.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2242-2244.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2242-2244.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2242-2244.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4056-4066.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4056-4066.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4056-4066.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4056- 4058.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4056-4058.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4056-4058.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 421-431.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 421-431.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs :421-423. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 421-423.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 421-431, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 421-431.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • sgRNA engineered synthetic guide ribonucleic acid
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence.
  • a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell and a tracr sequence.
  • the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5495-5502.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5495-5502.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of any one of SEQ ID NOs: 5495-5502.
  • the tracrRNA may comprise any of SEQ ID NOs: 5495-5502.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to any one of SEQ ID NOs: 5466-5467.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 5466-5467.
  • the sgRNA may comprise a sequence substantially identical to any one of SEQ ID NOs: 5466-5467.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2242- 2251.
  • RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2242- 2251.
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to any of SEQ ID NOs: 5578-5580 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 5578-5580.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2253-2481.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2253-2481.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2253-2481.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2253-2481.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2253-2481.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4067-4295.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4067-4295.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4067-4295.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4067- 4295.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4067-4295.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4067-4295.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 432-660.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 432-660.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 432-660.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 432-660.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 432-660, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 432-660.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5503.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5503.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5503.
  • the tracrRNA may comprise SEQ ID NO: 5503.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5468.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5468.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5468.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2253- 2481.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40,
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2482-2489.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2482-2489.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4296-4303.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4296-4303.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4056-4066.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 661-668. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 661-668.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 661-668, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 661-668.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence.
  • the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the system above may comprise two different guide RNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb)
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2482- 2489.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2490-2498.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2490-2498.
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2490-2498.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2490-2498.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2490-2498.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2490-2498.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4304-4312.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4304-4312.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4304-4312.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4304- 4312.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4304-4312.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4304-4312.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 669-677.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 669-677.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 669-677. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 669-677.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 669-677, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 669-677.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof: [00273]
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5504.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5504.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5504.
  • the tracrRNA may comprise SEQ ID NO: 5504.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2490- 2498.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic.
  • the organism may be fungal.
  • the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2499-2750.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2499-2750.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2499-2750.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2499-2750.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2499-2750.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4313-4564.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4313-4564.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4313-4564.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4313- 4564.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4067-4295.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4313-4564.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 678-929. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 678-929.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 678-929. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 678-929.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 678-929, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 678-929.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5505.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5505.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5505.
  • the tracrRNA may comprise SEQ ID NO: 5505.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5469.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5469.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5469.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2499-
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NO: 5581 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5581.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2751-2913.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2751-2913.
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2751-2913.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2751-2913.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2751-2913.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2751-2913.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4565-4727.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4565-4727.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4565-4727.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4565- 4727.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4565-4727.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4565-4727.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 930-1092.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 930-1092.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 930-1092. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 930-1092.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 930-1092, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 930-1092.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5506.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5506.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5506.
  • the tracrRNA may comprise SEQ ID NO: 5506.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5470.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5470.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5470.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2751- 2913.
  • RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2751- 2913.
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NO: 5582 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5582.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40,
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2914-3174.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 2914-3174.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2914-3174.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 2914-3174.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 2914-3174.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4728-4988.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4728-4988.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4728-4988.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4728- 4988.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4728-4988.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1093-1353.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1093-1353.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1093-1353.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 1093-1353, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1093-1353.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5507.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5507.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5507.
  • the tracrRNA may comprise SEQ ID NO: 5507.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5471.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5471.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5471.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 2914-
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NO: 5583 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5583.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40,
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3175-3300.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 3175-3300.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3175-3300.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 3175-3300.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 3175-3300.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4989-5146.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4989-5146.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4989-5146.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4989- 5146.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 4989-5146.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 4989-5146.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1354-1511.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1354-1511.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1354-1511.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1354-1511.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 1354-1511, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1354-1511.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5508.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5508.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5508.
  • the tracrRNA may comprise SEQ ID NO: 5508.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5472.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5472.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5472.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’ : a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lk
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 3175- 3300.
  • RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 3175- 3300.
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NOs: 5584 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 5584.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40,
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3331-3474.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 3331-3474.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3331-3474.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 3331-3474.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 3331-3474.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5147-5290.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 5147-5290.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 5147-5290.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5147- 5290.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 5147-5290.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 5147-5290.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1512-1655.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1512-1655.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1512-1655. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1512-1655.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 1512-1655, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1512-1655.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5509.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5509.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5509.
  • the tracrRNA may comprise SEQ ID NO: 5509.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5473.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5473.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5473.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 3331- 3474.
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NOs: 5585 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 5585.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40,
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3475-3568.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 3475-3568.
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 3475-3568.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3475-3568.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 3475-3568.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 3475-3568.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5291-5389.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 5291-5389.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 5291-5389.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5291- 5389.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 5291-5389.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 5291-5389.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1656-1755.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1656-1755.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1656-1755. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1656-1755.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 432-660, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1656-1755.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5510.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5510.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5510.
  • the tracrRNA may comprise SEQ ID NO: 5510.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5474.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5474.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5474.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 3475-
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NOs: 5586 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 5586.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • the present disclosure provides for an engineered nuclease system comprising (a) an endonuclease.
  • the endonuclease is a Cas endonuclease.
  • the endonuclease is a Type II, Class II Cas endonuclease.
  • the endonuclease may comprise a RuvC III domain, wherein said RuvC III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3569-3637.
  • the endonuclease may comprise a RuvC III domain, wherein the RuvC III domain has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about
  • the endonuclease may comprise a RuvC III domain, wherein the substantially identical to any one of SEQ ID NOs: 3569-3637.
  • the endonuclease may comprise a RuvC III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3569-3637.
  • the endonuclease may comprise a RuvC III domain having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of SEQ ID NOs: 3569-3637.
  • the endonuclease may comprise a RuvC III domain substantially identical to any one of SEQ ID NOs: 3569-3637.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5390-5460.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 5390-5460.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 5390-5460.
  • the endonuclease may comprise an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5390- 5460.
  • the endonuclease may comprise an HNH domain having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 5390-5460.
  • the endonuclease may comprise an HNH domain substantially identical to any one of SEQ ID NOs: 5390-5460.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1756-1826.
  • the endonuclease may be substantially identical to any one of SEQ ID NOs: 1756-1826.
  • the endonuclease may comprise a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1756-1826. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1756-1826.
  • the endonuclease may comprise a variant having one or more nuclear localization sequences (NLSs).
  • the NLS may be proximal to the N- or C-terminus of said endonuclease.
  • the NLS may be appended N-terminal or C-terminal to any one of SEQ ID NOs: 1756-1826, or to a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1756-1826.
  • the NLS may be an SV40 large T antigen NLS.
  • the NLS may be a c-myc NLS.
  • the NLS can comprise a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise a sequence substantially identical to any one of SEQ ID NOs: 5593-5608.
  • the NLS can comprise any of the sequences in Table 1 or a combination thereof:
  • sequence identity may be determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithm.
  • the sequence identity may be determined by the BLASTP algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and using a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
  • the system above may comprise (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease bearing a 5’ targeting region complementary to a desired cleavage sequence.
  • the 5’ targeting region may comprises a PAM sequence compatible with the endonuclease.
  • the 5’ most nucleotide of the targeting region may be G.
  • the 5’ targeting region may be 15-23 nucleotides in length.
  • the guide sequence and the tracr sequence may be supplied as separate ribonucleic acids (RNAs) or a single ribonucleic acid (RNA).
  • the guide RNA may comprise a crRNA tracrRNA binding sequence 3’ to the targeting region.
  • the guide RNA may comprise a tracrRNA sequence preceded by a 4-nucleotide linker 3’ to the crRNA tracrRNA binding region.
  • the sgRNA may comprise, from 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.
  • the tracr sequence may have a particular sequence.
  • the tracr sequence may have at least about 80% to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of a natural tracrRNA sequence.
  • the tracr sequence may have at least about 80% sequence identity to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5511.
  • the tracrRNA may have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least about 60-90 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5511.
  • at least about 60-90 e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90
  • the tracrRNA may be substantially identical to at least about 60-100 (e.g., at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90) consecutive nucleotides of SEQ ID NO: 5511.
  • the tracrRNA may comprise SEQ ID NO: 5511.
  • the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with the endonuclease may comprise a sequence having at least about 80% identity to SEQ ID NO: 5475.
  • the sgRNA may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 5475.
  • the sgRNA may comprise a sequence substantially identical to SEQ ID NO: 5475.
  • the system above may comprise two different sgRNAs targeting a first region and a second region for cleavage in a target DNA locus, wherein the second region is 3’ to the first region.
  • the system above may comprise a single- or double-stranded DNA repair template comprising from 5’ to 3’: a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 5’ to the first region, a synthetic DNA sequence of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb) nucleotides 3’ to the second region.
  • a first homology arm comprising a sequence of at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or lkb
  • the present disclosure provides a method for modifying a target nucleic acid locus of interest.
  • the method may comprise delivering to the target nucleic acid locus any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein.
  • the enzyme may form a complex with the at least one sgRNA, and upon binding of the complex to the target nucleic acid locus of interest, may modify the target nucleic acid locus of interest.
  • Delivering the enzyme to said locus may comprise transfecting a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise electroporating a cell with the system or nucleic acids encoding the system.
  • Delivering the nuclease to said locus may comprise incubating the system in a buffer with a nucleic acid comprising the locus of interest.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid locus may comprise genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the target nucleic acid locus may be within a cell.
  • the target nucleic acid locus may be in vitro.
  • the target nucleic acid locus may be within a eukaryotic cell or a prokaryotic cell.
  • the cell may be an animal cell, a human cell, bacterial cell, archaeal cell, or a plant cell.
  • the enzyme may induce a single or double-stranded break at or proximal to the target locus of interest.
  • the enzyme may be supplied as a nucleic acid containing an open reading frame encoding the enzyme having a RuvC III domain having at least about 75% (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) identity to any one of SEQ ID NOs: 3569-
  • the deoxyribonucleic acid (DNA) containing an open reading frame encoding said endonuclease may comprise a sequence substantially identical to SEQ ID NOs: 5587 or at variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 5587.
  • the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked.
  • the promoter may be a CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, or CaMKIIa promoter.
  • the endonuclease may be supplied as a capped mRNA containing said open reading frame encoding said endonuclease.
  • the endonuclease may be supplied as a translated polypeptide.
  • the at least one engineered sgRNA may be supplied as deoxyribonucleic acid (DNA) containing a gene sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the organism may be eukaryotic. In some cases, the organism may be fungal. In some cases, the organism may be human.
  • Metagenomic samples were collected from sediment, soil and animal.
  • Deoxyribonucleic acid (DNA) was extracted with a Zymobiomics DNA mini-prep kit and sequenced on an Illumina HiSeq® 2500. Samples were collected with consent of property owners. Additional raw sequence data from public sources included animal microbiomes, sediment, soil, hot springs, hydrothermal vents, marine, peat bogs, permafrost, and sewage sequences.
  • Metagenomic sequence data was searched using Hidden Markov Models generated based on known Cas protein sequences including type II Cas effector proteins. Novel effector proteins identified by the search were aligned to known proteins to identify potential active sites.
  • Example 2 A Discovery of an MG1 Family of CRISPR systems
  • Example 1 Analysis of the data from the metagenomic analysis of Example 1 revealed a new cluster of previously undescribed putative CRISPR systems initially comprising six members (MGl-1, MG1-2, MG1-3, MG1-4, MG1-5, and MG1-6 recorded as SEQ ID NOs: 5, 6, 1, 2, and 3 respectively).
  • This family is characterized by an enzyme bearing HNH and RuvC domains.
  • the RuvC domains of this family have a RuvC III portion having low homology to previously described Cas9 family members.
  • Example 2B Discovery of an MG2 Family of CRISPR systems
  • Example 1 Analysis of data from the metagenomic analysis of Example 1 revealed a new cluster of previously undescribed putative CRISPR systems comprising six members (MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6).
  • the corresponding protein and nucleic acid sequences for these new enzymes and exemplary subdomains are presented as SEQ ID NOs: 320, 322-325.
  • putative tracrRNA sequences were identified in the operon and are presented as SEQ ID NOs: 5490, 5492-5494, and 5538.
  • Example 2C Discovery of an MG3 Family of CRISPR systems
  • Example 1 Analysis of the data from the metagenomic analysis of Example 1 revealed a new previously undescribed putative CRISPR system: MG3-1.
  • the corresponding amino acid sequences for this new enzyme and its exemplary subdomains are presented as SEQ ID NOs:
  • FIGURE 8 A detailed domain-level alignment of the sequence versus Cas9 from Actinomyces naeslundii is depicted in FIGURE 8.
  • Example 2D - Discovery of MG4, 7, 14, 15, 16, 18, 21, 22, 23 Families of CRISPR systems
  • Analysis of the data from the metagenomic analysis of Example 1 revealed new clusters of previously undescribed putative CRISPR systems comprising 9 families of one member each (MG 4-5, MG7-2, MG14-1, MG15-1, MG16-2, MG18-1, MG21-1, MG22-1, MG23-1).
  • the corresponding protein and nucleic acid sequences for these new enzymes and their exemplary subdomains are presented as SEQ ID NOs: 432, 669, 678, 930, 1093, 1354, 1512, 1656, 1756.
  • a putative tracr containing sequence was identified for each family. These sequences are presented in the sequence listing as SEQ ID NOs: 5503-5511, respectively.
  • Motifs common to the nucleases of these sets of CRISPR systems are presented as SEQ ID NO: 5649 for MG4; SEQ ID NOs: 5650-5667 for MG14; 5668-5675 for MG15; SEQ ID NOs: 5676-5678 for MG16; SEQ ID NOs: 5679-5686 for MG18; SEQ ID NOs: 5687-5693 and SEQ ID NOs: 5674-5675 for MG21; SEQ ID NOs: 5694-5699 for MG22; and SEQ ID NOs: 5700-5717 for MG23.
  • cells bearing plasmids encoding any of the enzymes described herein and protospacer-targeting guide RNA are co-transformed with a plasmid library containing an antibiotic resistance gene, and a protospacer sequence flanked by a randomized PAM sequence. Plasmids containing functional PAMs are cleaved by the enzyme, leading to cell death. Deep-sequencing of the enzyme cleavage-resistant plasmid pool isolated from the surviving cells displays a set of depleted plasmids that contain functional cleavage-permitting PAMs.
  • PAM library in the form of DNA plasmid or concatemeric repeats is subjected to cleavage by the RNP complex (e.g., including the enzyme, tracrRNA and crRNA or the enzyme and hybrid sgRNA) assembled in vitro or in cell lysates. Resulting free DNA ends from successful cleavage events are captured by adapter ligation, followed by the PCR amplification of the PAM-sided products. Amplified library of functional PAMs is subjected to deep sequencing and PAMs licensing DNA cleavage are identified.
  • RNP complex e.g., including the enzyme, tracrRNA and crRNA or the enzyme and hybrid sgRNA
  • DNA/RNA sequences encoding (i) an ORF encoding codon-optimized enzyme under a cell-compatible promoter with a cell-compatible C-terminal nuclear localization sequence (e.g., SV40 NLS in the case of human cells) and a suitable polyadenylation signal (e.g., TK pA signal in the case of human cells); and (ii) an ORF encoding an sgRNA (having a 5’ sequence beginning with G followed by 20 nt of a complementary targeting nucleic acid sequence targeting genomic DNA followed by a corresponding compatible PAM identified via Example 3 and a 3 tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable
  • Polymerase III promoter (e.g., the U6 promoter in mammalian cells) are prepared. In some embodiments, these sequences are prepared on the same or separate plasmid vectors, which are transfected via a suitable technique into eukaryotic cells. In some embodiments, these sequences are prepared as separate DNA sequences, which are transfected or microinjected into cells. In some embodiments, these sequences are prepared as synthesized RNAs or in-vitro transcribed RNAs which are transfected or microinjected into cells. In some embodiments, these sequences are translated into proteins and transfected or microinjected into cells.
  • these sequences are prepared on the same or separate plasmid vectors, which are transfected via a suitable technique into eukaryotic cells. In some embodiments, these sequences are prepared as separate DNA sequences, which are transfected or microinjected into cells. In some embodiments, these sequences are prepared as synthesized RNAs or in-vitro transcribed RNAs which are transfected
  • (i) and (ii) are introduced into cells with a third repair nucleotide that encodes regions of the genome flanking the cleavage site of sizes 25 bp or larger, which will facilitate homology directed repair. Containing within these flanking sequences may be a single base pair mutation, a functional gene fragment, a foreign or native gene for expression, or several genes composing a biochemical pathway.
  • RNAs comprising a 5’ G followed by a 20 nt targeting sequence and PAM sequence, a tracrRNA binding region of a compatible crRNA, a GAAA linker, and a compatible tracrRNA are synthesized by suitable solid-phase RNA synthesis methods.
  • Recombinant enzymes and sgRNA are combined in a suitable cleavage buffer containing Mg2+ (e.g., 20 mM HEPES pH 7.5, 100 mM KC1, 5 mM MgCh, 1 mM DTT, 5% glycerol) and the reaction is initiated by introducing a target DNA including a sequence complementary to the targeting sequence and PAM sequence. Cleavage of the DNA is monitored by a suitable assay (e.g., agarose gel electrophoresis followed by ethidium bromide staining (or similarly acting DNA-intercalating agent) and UV visualization).
  • Mg2+ e.g., 20 mM HEPES pH 7.5, 100 mM KC1, 5 mM MgCh, 1 mM DTT, 5% glycerol
  • PAM sequences were determined by sequencing plasmids containing randomly- generated PAM sequences that could be cleaved by putative endonucleases expressed in an E. coli lysate-based expression system (myTXTL, Arbor Biosciences).
  • E. coli codon optimized nucleotide sequence was transcribed and translated from a PCR fragment under control of a T7 promoter.
  • a second PCR fragment with a tracr sequence under a T7 promoter and a minimal CRISPR array composed of a T7 promoter followed by a repeat-spacer-repeat sequence was transcribed in the same reaction.
  • Successful expression of the endonuclease and tracr sequence in the TXTL system followed by CRISPR array processing provided active in vitro CRISPR nuclease complexes.
  • a library of target plasmids containing a spacer sequence matching that in the minimal array followed by 8N mixed bases (putative PAM sequences) was incubated with the output of the TXTL reaction. After 1-3 hr, the reaction was stopped and the DNA was recovered via a DNA clean-up kit, e.g., Zymo DCC, AMPure XP beads, QiaQuick etc. Adapter sequences were blunt-end ligated to DNA with active PAM sequences that had been cleaved by the
  • DNA segments comprising active PAM sequences were then amplified by PCR with primers specific to the library and the adapter sequence.
  • the PCR amplification products were resolved on a gel to identify amplicons that corresponded to cleavage events.
  • the amplified segments of the cleavage reaction were also used as template for preparation of an NGS library. Sequencing this resulting library, which was a subset of the starting 8N library, revealed the sequences which contain the correct PAM for the active CRISPR complex.
  • Endonucleases were expressed as His-tagged fusion proteins from an inducible T7 promoter in a protease deficient A. coli B strain. Cells expressing the His-tagged proteins were lysed by sonication and the His-tagged proteins were purified by Ni-NTA affinity
  • Target DNAs containing spacer sequences and PAM sequences were constructed by DNA synthesis. A single representative PAM was chosen for testing when the PAM had degenerate bases .
  • the target DNAs comprised 2200 bp of linear DNA derived from a plasmid via PCR amplification with a PAM and spacer located 700 bp from one end. Successful cleavage resulted in fragments of 700 and 1500 bp.
  • cleavage buffer (10 mM Tris, 100 mM NaCl, 10 mM MgCh) with an excess of protein and RNA and incubated for 5 minutes to 3 hours, usually 1 hr.
  • the reaction was stopped via addition of RNAse A and incubation at 60 minutes.
  • the reaction was then resolved on a 1.2% TAE agarose gel and the fraction of cleaved target DNA is quantified in ImageLab software.
  • E. coli lacks the capacity to efficiently repair double-stranded DNA breaks. Thus, cleavage of genomic DNA can be a lethal event. Exploiting this phenomenon, endonuclease activity was tested in E. coli by recombinantly expressing an endonuclease and a tracrRNA in a target strain with spacer/target and PAM sequences integrated into its genomic DNA.
  • the PAM sequence is specific for the endonuclease being tested as determined by the methods described in Example 6.
  • sgRNA sequences were determined based upon the sequence and predicted structure of the tracrRNA. Repeat-anti -repeat pairings of 8-12 bp (generally lObp) were chosen, starting from the 5’ end of the repeat. The remaining 3’ end of the repeat and 5’ end of the tracrRNA were replaced with a tetraloop.
  • the tetraloop was GAAA, but other tetraloops can be used, particularly if the GAAA sequence is predicted to interfere with folding. In these cases, a TTCG tetraloop was used.
  • the MG Cas effector protein sequences were tested in two mammalian expression vectors: (a) one with a C-terminal SV40 NLS and a 2A-GFP tag, and (b) one with no GFP tag and two SV40 NLS sequences, one on the N-terminus and one on the C-terminus.
  • nucleotide sequences encoding the endonucleases were codon-optimized for expression in mammalian cells.
  • the corresponding single guide RNA sequence (sgRNA) with targeting sequence attached is cloned into a second mammalian expression vector.
  • the two plasmids are cotransfected into HEK293T cells.
  • 72 hr after co-transfection of the expression plasmid and a sgRNA targeting plasmid into HEK293T cells the DNA is extracted and used for the preparation of an NGS-library.
  • Percent NHEJ is measured via indels in the sequencing of the target site to demonstrate the targeting efficiency of the enzyme in mammalian cells. At least 10 different target sites were chosen to test each protein’s activity.
  • Synthetic single guide RNAs were designed based on the sequences and predicted structures of the tracrRNAs and are presented as SEQ ID NOs: 5461-5464.
  • the PAM sequence screen of Example 6 was repeated with the sgRNAs.
  • the results of this experiment are also presented in Table 2, which reveals that PAM specificity changed slightly when using sgRNAs.
  • Example 10 The method of Example 10 was used to demonstrate targeting and cleavage activity in mammalian cells. Open reading frames encoding the MG1-4 (protein SEQ ID NO: 5527) and MG1-6 (protein SEQ ID NO: 5529) sequences were cloned into 2 mammalian expression vectors, one with a C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB) and one with no GFP tag and 2 NLS sequences, one on the N-terminus and one on the C-terminus (E. coli pMG5- BB).
  • Open reading frames encoding the MG1-4 (protein SEQ ID NO: 5527) and MG1-6 (protein SEQ ID NO: 5529) sequences were cloned into 2 mammalian expression vectors, one with a C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB) and one with no GFP tag and 2 NLS sequences, one on the
  • MG1-6 the open reading frame was additionally codon-optimized for mammalian expression (SEQ ID NO: 5589) and cloned into the 2-NLS plasmid backbone (MG-16hs).
  • SEQ ID NO: 5589 codon-optimized for mammalian expression
  • MG-16hs 2-NLS plasmid backbone
  • the results of this experiment are shown in Figure 12.
  • the endonuclease expression vectors were cotransfected into HEK293T cells with a second vector for expressing a sgRNA (e.g., SEQ ID NOs: 5512 or 5515) with a tracr sequence specific for the endonuclease and a guide sequence selected from Tables 3-4. 72 hr after co-transfection the DNA was extracted and used for the preparation of an NGS-library.
  • a sgRNA e.g., SEQ ID NOs: 5512 or 5515
  • NHEJ remnants Cleavage activity was detected by the appearance of internal deletions (NHEJ remnants) proximal to the sequence of the target site. Percent NHEJ was measured via indels in the sequencing of the target site to demonstrate the targeting efficiency of the enzyme in mammalian cells and is presented in Figure 12.
  • Targeted endonuclease activity in bacterial cells [00404] In vivo activity of the MG2-7 endonuclease system with a sgRNA (endonuclease SEQ ID NO: 321; sgRNA SEQ ID NO: 5465) and an AGCGTAAG PAM sequence was confirmed using the method described in Example 9 . Transformed E. coli were plated in serial dilution, and the results (showing E. coli serial dilutions in the left panel and quantitated growth in the right panel) are presented in Figure 34. A substantial reduction in the growth of E. coli expressing on target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by the MG1-4 endonuclease in E. coli cells.
  • Synthetic single guide RNAs were designed based on the sequences and predicted structures of the tracrRNAs and are presented as SEQ ID NOs: 5466-5467.
  • the PAM sequence screen of Example 6 was repeated with the sgRNAs.
  • the results of this experiment are also presented in Table 6, which reveals that PAM specificity changed slightly when using sgRNAs.
  • Example 10 The method of Example 10 was used to demonstrate targeting and cleavage activity in mammalian cells.
  • Open reading frames encoding MG3-7 (protein SEQ ID NO: 422) was cloned into 2 mammalian expression vectors, one with a C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB) and one with no GFP tag and 2 NLS sequences, one on the N-terminus and one on the C-terminus (E. coli pMG5-BB).
  • the endonuclease expression vectors were cotransfected into HEK293T cells with a second vector for expressing the sgRNA above with a guide sequence selected from Table 7.
  • Targeted endonuclease activity in bacterial cells [00423] In vivo activity of the MG14-1 endonuclease system with a sgRNA (endonuclease SEQ ID NO: 678; sgRNA SEQ ID NO: 5469) and a GGCGGGGA PAM sequence was confirmed using the method described in Example 9 . Transformed E. coli were plated in serial dilution, and the results (showing E. coli serial dilutions in the left panel and quantitated growth in the right panel) are presented in Figure 35. A substantial reduction in the growth of E. coli expressing on target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by the MG1-4 endonuclease in E. coli cells.
  • Targeted endonuclease activity in bacterial cells [00430] In vivo activity of the MG15-1 endonuclease system with a sgRNA (endonuclease SEQ ID NO: 930; sgRNA SEQ ID NO: 5470) and a GGGTCAAA PAM sequence was confirmed using the method described in Example 9 . Transformed E. coli were plated in serial dilution, and the results (showing E. coli serial dilutions in the left panel and quantitated growth in the right panel) are presented in Figure 35. A substantial reduction in the growth of E. coli expressing on target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by the MG1-4 endonuclease in E. coli cells.
  • Systems of the present disclosure may be used for various applications, such as, for example, nucleic acid editing (e.g., gene editing), binding to a nucleic acid molecule (e.g., sequence-specific binding).
  • nucleic acid editing e.g., gene editing
  • binding to a nucleic acid molecule e.g., sequence-specific binding
  • Such systems may be used, for example, for addressing (e.g., removing or replacing) a genetically inherited mutation that may cause a disease in a subject, inactivating a gene in order to ascertain its function in a cell, as a diagnostic tool to detect disease-causing genetic elements (e.g.

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CN202080028787.6A CN113728098A (zh) 2019-02-14 2020-02-14 具有ruvc结构域的酶
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EP20756259.6A EP3924482A4 (en) 2019-02-14 2020-02-14 ENZYMES WITH RUVC DOMAINS
KR1020217028874A KR102623312B1 (ko) 2019-02-14 2020-02-14 Ruvc 도메인이 존재하는 효소
CA3130135A CA3130135C (en) 2019-02-14 2020-02-14 Enzymes with RuVC domains
AU2020223370A AU2020223370B2 (en) 2019-02-14 2020-02-14 Enzymes with RuvC domains
JP2021547336A JP2022520428A (ja) 2019-02-14 2020-02-14 Ｒｕｖｃドメインを有する酵素
KR1020247000311A KR20240007322A (ko) 2019-02-14 2020-02-14 Ruvc 도메인이 존재하는 효소
MX2021009886A MX2021009886A (es) 2019-02-14 2020-02-14 Enzimas con dominios ruvc.
US16/917,837 US10982200B2 (en) 2019-02-14 2020-06-30 Enzymes with RuvC domains
US16/917,838 US10913941B2 (en) 2019-02-14 2020-06-30 Enzymes with RuvC domains
GB2219441.9A GB2617659B (en) 2020-02-14 2020-07-13 Enzymes with RuvC domains
GB2010770.2A GB2594339B (en) 2020-02-14 2020-07-13 Enzymes with RUVC domains
US17/193,173 US12024727B2 (en) 2019-02-14 2021-03-05 Enzymes with RuvC domains
US17/402,426 US20220220460A1 (en) 2019-02-14 2021-08-13 Enzymes with ruvc domains
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US18/668,543 US20240344045A1 (en) 2019-02-14 2024-05-20 Enzymes with ruvc domains
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