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WO2024083135A1 - Polypeptides iscb et leurs utilisations - Google Patents

Polypeptides iscb et leurs utilisations Download PDF

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Publication number
WO2024083135A1
WO2024083135A1 PCT/CN2023/125069 CN2023125069W WO2024083135A1 WO 2024083135 A1 WO2024083135 A1 WO 2024083135A1 CN 2023125069 W CN2023125069 W CN 2023125069W WO 2024083135 A1 WO2024083135 A1 WO 2024083135A1
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Prior art keywords
iscb
polypeptide
amino acid
sequence
domain
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Inventor
Hainan ZHANG
Yingsi ZHOU
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Huidagene Therapeutics Co Ltd
Huidagene Therapeutics Singapore Pte Ltd
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Huidagene Therapeutics Co Ltd
Huidagene Therapeutics Singapore Pte Ltd
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Publication of WO2024083135A1 publication Critical patent/WO2024083135A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]

Definitions

  • the disclosure contains a Sequence Listing XML file which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.
  • Said XML copy created on October 17, 2023, by software “WIPO Sequence” according to WIPO Standard ST. 26, is named HEP010PCT. xml, and is 201, 112 bytes in size.
  • IscB could be a nucleic acid programable DNA endonuclease that has a relatively small size, making it suitable for delivery. It would be desired to discover and develop IscB polypeptides for various applications, e.g., double-strand cleavage, base editing, prime editing.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid substitution at:
  • x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid substitution at:
  • x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid deletion at one or more (or all) amino acid residues of the RuvC I domain (e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain) , the RuvC II domain (e.g., one or more (or all) amino acid residues of the motif ExxxF in the RuvC I domain) , and/or the RuvC III domain (e.g., one or more (or all) amino acid residues of the motif HxxDA in the RuvC I domain) of a reference IscB polypeptide; wherein x is any amino acid residue.
  • the RuvC I domain e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain
  • the RuvC II domain e.g., one or more (or all) amino acid residues of the motif ExxxF in the RuvC I domain
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid deletion at one or more (or all) amino acid residues of the RuvC I domain (e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain) , the RuvC II domain (e.g., one or more (or all) amino acid residues of the motif ExxxF in the RuvC I domain) , and/or the RuvC III domain (e.g., one or more (or all) amino acid residues of the motif HxxDA in the RuvC I domain) of the reference IscB polypeptide, thereby generating the engineered IscB polypeptide; wherein x is any amino acid residue.
  • the RuvC I domain e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain
  • the RuvC II domain e
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid substitution at H 1 , N 1 , H 2 , H 3 , N 2 and/or K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide;
  • x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid substitution at H 1 , N 1 , H 2 , H 3 , N 2 and/or K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of the reference IscB polypeptide, thereby generating the engineered IscB polypeptide;
  • x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid deletion at one or more (or all) amino acid residues of the HNH domain (e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, wherein x is any amino acid residue) of a reference IscB polypeptide.
  • the HNH domain e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, wherein x is any amino acid residue
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid deletion at one or more (or all) amino acid residues of the HNH domain (e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, wherein x is any amino acid residue) of the reference IscB polypeptide, thereby generating the engineered IscB polypeptide.
  • an amino acid deletion at one or more (or all) amino acid residues of the HNH domain (e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, where
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • an engineered IscB polypeptide comprising:
  • x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide (1) has low or no endonuclease activity and (2) has low or no nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide:
  • x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide.
  • the engineered IscB polypeptide has low or no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the engineered IscB polypeptide has low or no nickase activity.
  • the engineered IscB polypeptide (1) has low or no endonuclease activity and (2) has low or no nickase activity.
  • the amino acid substitution comprises or is a non-conservative amino acid substitution.
  • amino acid substitution at D of motif DxG in the RuvC I domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at G of motif DxG in the RuvC I domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at H of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at D of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution
  • amino acid substitution at K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the non-conservative amino acid substitution is a non-conservative amino acid substitution with A.
  • the amino acid substitution comprises or is a conservative amino acid substitution.
  • amino acid substitution at D of motif DxG in the RuvC I domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at G of motif DxG in the RuvC I domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at H of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at D of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution
  • amino acid substitution at K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the conservative amino acid substitution is a conservative amino acid substitution with A
  • the amino acid substitution comprises or is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid. In some embodiments, the amino acid substitution comprises or is an amino acid substitution with a non-polar amino acid, such as, A.
  • amino acid substitution at D of motif DxG in the RuvC I domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at G of motif DxG in the RuvC I domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at H of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at D of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid;
  • amino acid substitution at N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid; and/or
  • amino acid substitution at K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) ; wherein the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) ; wherein the positively charged amino acid is selected from the group consisting of Lysine (Lys/K)
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the reference IscB polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 26-91 and 158, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 26-91 and 158.
  • D of motif DxG in the RuvC I domain is at a position of the reference IscB polypeptide corresponding to position D61 of SEQ ID NO: 29;
  • G of motif DxG in the RuvC I domain is at a position of the reference IscB polypeptide corresponding to position G63 of SEQ ID NO: 29;
  • H of motif HxxDA in the RuvC III domain is at a position of the reference IscB polypeptide corresponding to position H340 of SEQ ID NO: 29;
  • H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position H248 of SEQ ID NO: 29;
  • N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position N262 of SEQ ID NO: 29;
  • H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position H271 of SEQ ID NO: 29;
  • H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position H275 of SEQ ID NO: 29;
  • N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position N236 of SEQ ID NO: 35;
  • the engineered IscB polypeptide has a sequence identity of at least about 50% (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%) to the amino acid sequence of the reference IscB polypeptide.
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the engineered IscB polypeptide comprises (1) an amino acid substitution of D61A, D61E, D61Q, D61R, G63A, G63E, G63Q, G63R, E193A, E193D, E193Q, E193R, F197A, F197E, F197Q, F197R, H340A, H340E, H340Q, H340R, D343A, D343E, D343Q, D343R, A344G, A344E, A344Q, and/or A344R; (2) an amino acid substitution of H248A, H248R, H271A, H271R, H275A, and/or H275R; or (3) both (1) and (2) .
  • the engineered IscB polypeptide comprises an amino acid substitution of D61A.
  • the engineered IscB polypeptide comprises an amino acid substitution of D61A and H248A.
  • the engineered IscB polypeptide comprises SEQ ID NO: 170 or 171 or a N-terminal truncation of SEQ ID NO: 170 or 171 without N-terminal Methionine (M) .
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and the engineered IscB polypeptide comprises (1) an amino acid substitution of D60A, D60E, D60Q, D60R, G62A, G62E, G62Q, G62R, E190A, E190D, E190Q, E190R, F194A, F194E, F194Q, F194R, H333A, H333E, H333Q, H333R, D336A, D336E, D336Q, D336R, A337G, A337E, A337Q, and/or A337R; (2) an amino acid substitution of H244A, H244R, H267A, H267R, H271A, and/or H271R; or (3) both (1) and (2) .
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 89 or 158
  • the engineered IscB polypeptide comprises (1) an amino acid substitution of D61A, D61E, D61Q, D61R, G63A, G63E, G63Q, G63R, E193A, E193D, E193Q, E193R, F197A, F197E, F197Q, F197R, H340A, H340E, H340Q, H340R, D343A, D343E, D343Q, D343R, A344G, A344E, A344Q, and/or A344R; (2) an amino acid substitution of H247A; or (3) both (1) and (2) .
  • the engineered IscB polypeptide comprises any one of SEQ ID NOs: 164-166 or a N-terminal truncation of any one of SEQ ID NOs: 164-166 without N-terminal Methionine (M) .
  • the reference IscB polypeptide is CRISPR-associated.
  • the disclosure provides an IscB polypeptide:
  • the engineered IscB polypeptide or the IscB polypeptide is fused to a functional domain, e.g., a base editing domain.
  • the disclosure provides a system comprising:
  • a guide nucleic acid or a polypeptide encoding the guide nucleic acid comprising:
  • the scaffold sequence has substantially the same secondary structure as the secondary structure of any one of SEQ ID NOs: 92-157, and/or comprises a polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%) to the polynucleotide sequence of any one of SEQ ID NOs: 92-157.
  • the engineered IscB polypeptide, the reference IscB polypeptide, or the IscB polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 26-91, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 26-91, and the scaffold sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 92-157, or a polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the engineered IscB polypeptide or the IscB polypeptide is capable of targeting a target DNA (e.g., a dsDNA) , such as, a eukaryotic DNA, a DNA in a eukaryotic cell.
  • a target DNA e.g., a dsDNA
  • the disclosure provides a polynucleotide encoding the engineered IscB polypeptide or the IscB polypeptide of the disclosure.
  • the disclosure provides a vector comprising the polynucleotide of the disclosure.
  • the disclosure provides a method of modifying a target DNA, comprising contacting the target DNA with the system of the disclosure or the vector of the disclosure, wherein the guide sequence is capable of hybridizing to a target sequence of the target DNA, wherein the target DNA is modified by the system.
  • the disclosure provides a cell comprising the engineered IscB polypeptide or the IscB polypeptide of the disclosure, the system of the disclosure, or the vector of the disclosure; optionally, wherein the cell is a eukaryotic cell.
  • FIG. 1 shows schematic exemplary constructs of expression and reporter plasmids for the evaluation of endonuclease and nickase activities of IscB polypeptides.
  • FIG. 2 is a histogram showing the endonuclease activities of the mutants of HISCB016 compared with NT and WT, in which a substitution with A ( “X to A” ) or R ( “X to R” ) was introduced.
  • FIG. 3 is a histogram showing the nickase activities of the mutants of HISCB016 compared with NT and WT, in which a substitution with A ( “X to A” ) or R ( “X to R” ) was introduced.
  • FIG. 4 is a histogram showing the endonuclease activities of the mutants of HISCB017 compared with NT and WT, in which a substitution with A ( “X to A” ) or R ( “X to R” ) was introduced.
  • FIG. 5 is a histogram showing the nickase activities of the mutants of HISCB017 compared with NT and WT, in which a substitution with A ( “X to A” ) or R ( “X to R” ) was introduced.
  • FIG. 6 is a histogram showing the endonuclease activities of the mutants of OgeuIscB or enOgeuIscB compared with NT (the right column in each pair) and REF.
  • FIG. 7 is a histogram showing the nickase activities of the mutants of OgeuIscB or enOgeuIscB compared with NT (the right column in each pair) and REF.
  • FIG. 8 shows the domain organization of IscB. P1D, P1 interaction domain; TID, TAM-interaction domain. RuvC domain is separated into three segments: RuvC I, II, and III.
  • FIG. 9 shows the predicted domain origination of HISCB016 and the amino acid residue at the boundary between two domains.
  • Each amino acid residue at the boundary is assigned to the domain on its N’ terminal.
  • the amino acid residue at position 86 is assigned to RuvC I domain instead of Bridge Helix.
  • FIG. 10 shows the predicted domain origination of HISCB017 and the amino acid residue at the boundary between two domains.
  • Each amino acid residue at the boundary is assigned to the domain on its N’ terminal.
  • the amino acid residue at position 85 is assigned to RuvC I domain instead of Bridge Helix.
  • FIG. 11 illustrates an exemplary target DNA, and an exemplary IscB system comprising (1) an exemplary guide nucleic acid comprising a guide sequence and a scaffold sequence and (2) an exemplary IscB polypeptide.
  • IscB as a nucleic acid programmable DNA endonuclease similar to Cas9 and Cas12, is capable of binding to a DNA (e.g., a dsDNA) as guided by a guide nucleic acid (e.g., a guide RNA) comprising a guide sequence targeting the DNA.
  • IscB may be associated with the guide nucleic acid (e.g., a guide RNA) , which localizes /targets the IscB to a DNA that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the guide sequence of a guide RNA) .
  • the guide nucleic acid “programs” the IscB to localize and bind to the DNA. Binding of the IscB to the DNA enables the IscB or a construct comprising the IscB to access to and function on the DNA.
  • IscB is discussed in Altae-Tran H, Kannan S, Demircioglu FE et al. .
  • the widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. SCIENCE 2021; 374 (6563) : 57-65 (and all its supplementary materials) ; Kato, K., Okazaki, S., Kannan, S. et al.
  • the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the IscB, and a guide sequence that is intentionally designed to be responsible for hybridizing to a target sequence of the DNA, thereby guiding the complex comprising the IscB and the guide nucleic acid to the DNA such that the IscB is indirectly bound to the DNA.
  • IscB nucleic acid programmable DNA binding protein
  • an exemplary dsDNA is depicted to comprise a 5’ to 3’ single DNA strand and a 3’ to 5’ single DNA strand.
  • An exemplary guide nucleic acid is depicted to comprise a guide sequence and a scaffold sequence.
  • the guide sequence is designed to hybridize to a part of the 3’ to 5’ single DNA strand, and so the guide sequence “targets” that part.
  • the 3’ to 5’ single DNA strand is referred to as a “target strand (TS) ” of the dsDNA
  • the opposite 5’ to 3’ single DNA strand is referred to as a “nontarget strand (NTS) ” of the dsDNA.
  • target sequence That part of the target strand based on which the guide sequence is designed and to which the guide sequence may hybridize is referred to as a “target sequence”
  • protospacer sequence the opposite part on the nontarget strand corresponding to that part is referred to as the “protospacer sequence” , which is typically 100%(fully) reversely complementary to the target sequence, if there is no intentional or unintentional mismatch.
  • nucleic acid sequence e.g., a DNA sequence
  • a nucleic acid sequence is written in 5’ to 3’ direction /orientation unless explicitly indicated otherwise.
  • a DNA sequence of ATGC it is usually understood as 5’-ATGC-3’ unless otherwise indicated. Its reverse sequence is 5’-CGTA-3’. Its fully complementary sequence is 5’-TACG-3’. Its fully reverse complementary sequence is 5’-GCAT-3’. Note that the fully complementary sequence usually does not have the ability to base-pair /hybridize with the original sequence.
  • the double-strand sequence of a dsDNA may be represented with the sequence of its 5’ to 3’ single DNA strand conventionally written in 5’ to 3’ direction /orientation unless otherwise indicated.
  • the dsDNA may be simply represented as 5’-ATGC-3’. 5’-----ATGC -----3’ 3’-----TACG -----5’
  • either the 5’ to 3’ single DNA strand or the 3’ to 5’ single DNA strand of a dsDNA can be a nontarget strand from which a protospacer sequence is selected.
  • the strand on which the target nucleotide (e.g., deoxyribonucleotide dA) to be edited is located is termed as an edited strand, and the opposite strand is termed as a non-edited strand.
  • the nontarget strand is the edited strand
  • the target strand is the non-edited strand.
  • the 5’ to 3’ single DNA strand of the gene is the sense strand
  • the 3’ to 5’ single DNA strand of the gene is the antisense strand.
  • either the sense strand or the antisense strand of a gene can be a nontarget strand from which a protospacer sequence is selected.
  • the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-AUGC-3’ that is fully reversely complementary to the 3’ to 5’ strand of the dsRNA (3’-TACG-5’) , which would be set forth in ATGC in the electric sequence listing and marked as an RNA sequence according to WIPO standard ST.
  • the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-GCAU-3’ that is fully reversely complementary to the 5’ to 3’ strand of the dsDNA (5’-ATGC-3’) , which would be set forth in GCAT in the electric sequence listing and marked as an RNA sequence according to WIPO standard ST. 26.
  • the guide sequence of a guide nucleic acid is fully reversely complementary to the target sequence and the target sequence is fully reversely complementary to the protospacer sequence
  • the guide sequence is identical to the protospacer sequence except for the difference between the U in the guide sequence due to its RNA nature and the corresponding T in the protospacer sequence due to its DNA nature.
  • symbol “t” is used to denote both T in DNA and U in RNA (See “Table 1: List of nucleotides symbols” , the definition of symbol “t” is “thymine in DNA/uracil in RNA (t/u) ” ) .
  • such a guide sequence could be set forth in the same sequence as a corresponding protospacer sequence.
  • a single SEQ ID NO in the electronic sequence listing can be used to denote both such guide sequence and protospacer sequence, although such a single SEQ ID NO may be marked as either DNA or RNA in the electronic sequence listing.
  • SEQ ID NO that sets forth a protospacer /guide sequence it refers to either a protospacer sequence that is a DNA sequence or a guide sequence that is an RNA sequence depending on the context, no matter whether it is marked as a DNA or an RNA in the electronic sequence listing.
  • IscB contains a RuvC nuclease domain separated into three segments: RuvC I, II, and III domains, which is responsible for single-strand cleavage at the nontarget strand of a dsDNA, and a HNH nuclease domain, which is responsible for single-strand cleavage at the target strand of a dsDNA, together leading to double-strand cleavage of the dsDNA.
  • nucleic acid As used herein, the terms “nucleic acid” , “nucleic acid molecule” , or “polynucleotide” are used interchangeably. They refer to a polymer of deoxyribonucleotides or ribonucleotides or their mixtures of any length in either single-or double-stranded form, and, unless otherwise stated, encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. The terms encompass nucleic acid-like structures with synthetic backbones, as well as amplification products. DNAs and RNAs are both polynucleotides.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) , nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O (6) -methylguanine, and 2-thiocytidine) , chemically modified bases
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • fusion protein refers to a protein created through the joining of two or more originally separate proteins, or portions thereof.
  • a linker may be present between each protein.
  • heterologous in reference to polypeptide domains, refers to the fact that the polypeptide domains do not naturally occur together (e.g., in the same polypeptide) .
  • a polypeptide domain from one polypeptide may be fused to a polypeptide domain from a different polypeptide.
  • the two polypeptide domains would be considered “heterologous” with respect to each other, as they do not naturally occur together.
  • heterologous in reference to nucleotide sequences, refers to the fact that the nucleotide sequences do not naturally occur together (e.g., in the same polynucleotide) .
  • a guide nucleic acid generated by the hand of man a guide sequence intentionally designed to target a human gene locus may be fused to a scaffold sequence from a microorganism. The two nucleotide sequences would be considered “heterologous” with respect to each other, as they do not naturally occur together.
  • guide nucleic acid refers to a nucleic acid-based molecule capable of forming a complex with an IscB polypeptide (e.g., via a scaffold sequence of the guide nucleic acid) , and comprises a sequence (e.g., a guide sequences) that is sufficient to hybridize to a target DNA and guides the complex to the target DNA, which includes but is not limited to RNA-based molecules, e.g., a guide RNA.
  • guide RNA (gRNA) ” RNA guide
  • gRNA guide RNA
  • RNA guide is used interchangeably.
  • the term “guide sequence” is used interchangeably with the term “spacer sequence” .
  • the term “complex” refers to a grouping of two or more molecules.
  • the complex comprises a polypeptide and a nucleic acid interacting with (e.g., binding to, coming into contact with, adhering to) one another.
  • the term “complex” can refer to a grouping of a guide nucleic acid and a polypeptide (e.g., an IscB polypeptide) .
  • the term “complex” can refer to a grouping of a guide nucleic acid, a polypeptide (e.g., an IscB polypeptide) , and a target DNA.
  • the term “activity” refers to a biological activity.
  • the activity includes enzymatic activity, e.g., catalytic ability of an effector.
  • the activity can include nuclease activity, e.g., DNA nuclease activity, dsDNA endonuclease activity, guide sequence-specific (on-target) dsDNA endonuclease activity, guide sequence-specific (on-target) ssDNA endonuclease activity (the ssDNA endonuclease activity is also termed as nickase activity herein) , guide sequence-independent (off-target) dsDNA endonuclease activity, base editing activity.
  • nuclease activity e.g., DNA nuclease activity, dsDNA endonuclease activity, guide sequence-specific (on-target) dsDNA endonuclease activity, guide sequence-specific (on-target) ssDNA endonuclea
  • base excising domain and “base excising protein” are used interchangeably to refer to a protein capable of excising a base from a deoxyribonucleotide.
  • a non-limiting example of the base excising protein is DNA glycosylase, which recognizes and removes a base from a deoxyribonucleotide by cleaving the base–sugar (N-glycosylic) bond, thereby excising the base from the deoxyribonucleotide (see A. C. Drohat, A. Maiti, DNA Glycosylases: Mechanisms, Encyclopedia of Biological Chemistry (Second Edition, Academic Press, 2013) .
  • the hypoxanthine excising domain functions to excise the hypoxanthine formed in situ by the adenine deamination domain of the base editor (i.e., excision after deamination) .
  • the activity of the base excising domain of the disclosure may not rely on the outcome of base deamination and may be independent of base deamination, which means the excision of the base may occur without deamination of the base before excision.
  • cleavage refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or cohesive ends.
  • cleaving a nucleic acid or “modifying a nucleic acid” may overlap. Modifying a nucleic acid includes not only modification of a mononucleotide but also insertion or deletion of a nucleic acid fragment.
  • on-target refers to binding, cleavage, editing, or otherwise modifying of an intended or expected region of a DNA, for example, by a base editor.
  • off-target refers to binding, cleavage, editing, or otherwise modifying of an unintended or unexpected region of a DNA, for example, by a base editor.
  • a region of a DNA is an off-target region when it differs from the region of the DNA intended or expected to be bound, cleaved edited, or otherwise modified by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.
  • RNA sequence As used herein, if a DNA sequence, for example, 5’-ATGC-3’ is transcribed to an RNA sequence, with each dT (deoxythymidine, or “T” for short) in the primary sequence of the DNA sequence replaced with a U (uridine) and each dA (deoxyadenosine, or “A” for short) , dG (deoxyguanosine, or “G” for short) , and dC (deoxycytidine, or “C” for short) replaced with A (adenosine) , G (guanosine) , and C (cytidine) , respectively, for example, 5’-AUGC-3’, it is said in the disclosure that the DNA sequence “encodes” the RNA sequence.
  • protospacer adjacent motif and “target adjacent motif (TAM) ” refer to a short sequence (or a motif) adjacent to a protospacer sequence on the nontarget strand of a dsDNA recognizable by an IscB polypeptide or a complex comprising an IscB polypeptide and a guide nucleic acid.
  • the PAM or TAM is immediately 3’ to a protospacer sequence.
  • adjacent includes instances wherein there is no nucleotide between the protospacer sequence and the PAM and also instances wherein there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the protospacer sequence and the PAM.
  • a “immediately adjacent (to) ” B, A “immediately 5’ to” B, and A “immediately 3’ to” B mean that there is no nucleotide between A and B.
  • the guide sequence is so designed to be substantially capable of hybridizing to a target sequence.
  • the term “hybridize” refers to a reaction in which one or more polynucleotide sequences react to form a complex that is stabilized via hydrogen bonding between the bases of the one or more polynucleotide sequences. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • a polynucleotide sequence capable of hybridizing to a given polynucleotide sequence is referred to as the “complement” of the given polynucleotide sequence.
  • the hybridization of a guide sequence and a target sequence is so stabilized to permit an IscB polypeptide that is complexed with a guide nucleic acid comprising the guide sequence or a function domain (e.g., a deaminase domain) associated (e.g., fused) with the IscB polypeptide to act (e.g., cleave, deaminize) at or near the target sequence or its complement (e.g., a sequence of a target DNA or its complement) .
  • a function domain e.g., a deaminase domain
  • the target sequence or its complement e.g., a sequence of a target DNA or its complement
  • the guide sequence is reversely complementary to a target sequence.
  • reverse complementary refers to the ability of nucleobases of a first polynucleotide sequence, such as a guide sequence, to base pair with nucleobases of a second polynucleotide sequence, such as a target sequence, by traditional Watson-Crick base-pairing. Two reverse complementary polynucleotide sequences are able to non-covalently bind under appropriate temperature and solution ionic strength conditions.
  • a first polynucleotide sequence (e.g., a guide sequence) comprises 100% (fully) reverse complementarity to a second nucleic acid (e.g., a target sequence) .
  • a first polynucleotide sequence (e.g., a guide sequence) is reverse complementary to a second polynucleotide sequence (e.g., a target sequence) if the first polynucleotide sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%complementarity to the second nucleic acid (i.e., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
  • the term “substantially complementary” refers to a polynucleotide sequence (e.g., a guide sequence) that has a certain level of complementarity to a second polynucleotide sequence (e.g., a target sequence) (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the guide sequence can base-pair with the polynucleotide sequence of the target sequence, or at most 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotides of the guide sequence mismatch the nucleotides of the target sequence) .
  • the level of complementarity is such that the first polynucleotide sequence (e.g., a guide sequence) can hybridize to the second polynucleotide sequence (e.g., a target sequence) with sufficient affinity to permit an IscB polypeptide that is complexed with the first polynucleotide sequence or a nucleic acid comprising the first polynucleotide sequence or a function domain (e.g., a deaminase domain) associated (e.g., fused) with the IscB polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement (e.g., a sequence of a target DNA or its complement) .
  • a function domain e.g., a deaminase domain
  • a guide sequence that is substantially complementary to a target sequence has 100%or less than 100%complementarity to the target sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%complementarity to the target sequence, and/or has at most 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotide mismatches from the target sequence.
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acids (e.g., DNA and/or RNA) and/or between polypeptides.
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical.
  • Calculation of the percent identity of two nucleic acids or polypeptides can be performed by aligning the two sequences for optimal comparison purpose (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes) .
  • the length of a sequence aligned for comparison purpose is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100%of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • nucleic acids or polypeptides may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.
  • sequence identity is calculated by global alignment, for example, using the Needleman-Wunsch algorithm and an online tool at ebi. ac. uk/Tools/psa/emboss_needle/.
  • sequence identity is calculated by local alignment, for example, using the Smith-Waterman algorithm and an online tool at ebi. ac. uk/Tools/psa/emboss_water/.
  • variant refers to an entity that shows significant structural identity with a reference entity (e.g., a wild-type sequence) but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements.
  • a polypeptide may have a characteristic sequence element comprising a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function;
  • a nucleic acid may have a characteristic sequence element comprising a plurality of nucleotide residues having designated positions relative to one another in linear or three-dimensional space.
  • a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc. ) covalently attached to the polypeptide backbone.
  • a variant polypeptide shows an overall sequence identity with a reference polypeptide (e.g., a nuclease described herein) that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%.
  • a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide, for example, an IscB nickase as a variant of a reference IscB polypeptide does not share an active RuvC domain or an active HNH domain with the reference IscB polypeptide.
  • the reference polypeptide has one or more biological activities.
  • a variant polypeptide shares one or more of the biological activities of the reference polypeptide, e.g., nuclease activity.
  • a variant polypeptide lacks one or more of the biological activities of the reference polypeptide, for example, an IscB nickase as a variant of a reference IscB polypeptide does not share the endonuclease activity of the reference IscB polypeptide.
  • a variant polypeptide shows a reduced level of one or more biological activities (e.g., nuclease activity, e.g., off-target nuclease activity) as compared with the reference polypeptide.
  • a polypeptide of interest is considered to be a “variant” of a reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the reference polypeptide but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%of the residues in the variant are substituted as compared with the reference polypeptide.
  • a variant has about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a reference polypeptide.
  • a variant has a very small number (e.g., fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues that participate in a particular biological activity) .
  • a variant has not more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the reference polypeptide.
  • any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues.
  • the reference polypeptide is a wild type polypeptide.
  • a variant of a polynucleotide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of a polynucleotide may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • nucleic acid or polypeptide As used herein, the terms “non-naturally occurring” and “engineered” are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid or a polypeptide, it is meant that the nucleic acid or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.
  • a “conservative substitution” refers to a substitution of an amino acid made among amino acids within one of the following four groups:
  • non-polar amino acids including Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ;
  • polar amino acids including Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) ; and
  • Lysine Lys/K
  • Arginine Arg/R
  • Histidine Histidine
  • wild type has the meaning commonly understood by those skilled in the art to mean a typical form of an organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.
  • upstream and downstream refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid. “Upstream” and “downstream” relate to the 5’ to 3’ direction, respectively, in which transcription occurs.
  • the first sequence is upstream of the second sequence when the 3’ end of the first sequence is on the left side of the 5’ end of the second sequence, and the first sequence is downstream of the second sequence when the 5’ end of the first sequence is on the right side of the 3’ end of the second sequence.
  • a promoter is usually at the upstream of a sequence under the regulation of the promoter; and on the other hand, a sequence under the regulation of a promoter is usually at the downstream of the promoter.
  • regulatory element refers to a DNA sequence that controls or impacts one or more aspects of transcription and/or expression and is intended to include promoters, enhancers, silencers, termination signals, internal ribosome entry sites (IRES) , and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences) .
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) . Regulatory elements may also direct expression in a time-dependent manner, e.g., in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory element “operably linked” to a functional element is associated in such a way that transcription, expression, and/or activity of the functional element is achieved under conditions compatible with the regulatory element.
  • “operably linked” regulatory elements are contiguous (e.g., covalently linked) with the functional elements of interest; in some embodiments, regulatory elements act in trans to or otherwise at a distance from the functional elements of interest.
  • the “cell” is understood to refer not only to a particular individual cell, but to the progeny or potential progeny of the cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term.
  • in vivo means to inside the body of an organism
  • ex vivo or in vitro means outside the body of an organism.
  • the term “treat” , “treatment” , or “treating” is an approach for obtaining beneficial or desired results including clinical results.
  • the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, stabilizing a disease (e.g., delaying the worsening of a disease) , delaying the spread (e.g., metastasis) of a disease, delaying the recurrence of a disease, reducing recurrence rate of a disease, delay or slowing the progression of a disease, ameliorating a disease state, providing a remission (partial or total) of a disease, decreasing the dose of one or more other medications required to treat a disease, delaying the progression of a disease, increasing the quality of life, and prolonging survival.
  • a reduction of pathological consequence of a disease such as cancer
  • disease includes the terms “disorder” and “condition” and is not limited to those specific diseases that have been medically or clinically defined.
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method may be used to treat cancer of types other than X.
  • the term “and/or” in a phrase such as “A and/or B” is intended to mean either or both of the alternatives, including both A and B, A or B, A (alone) , and B (alone) .
  • the term “and/or” in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .
  • a numerical range includes the end values of the range, and each specific value within the range, for example, “16 to 100 nucleotides” includes 16 nucleotides and 100 nucleotides, and each specific value between 16 and 100, e.g., 17, 23, 34, 52, 78.
  • the term “reference IscB polypeptide” is used in the context of designing and developing a new IscB polypeptide based on an original IscB polypeptide (e.g., a wild-type IscB polypeptide) , for example, the original IscB polypeptide is mutated to generate the new IscB polypeptide. In that case, the original IscB polypeptide is a reference of the new IscB polypeptide and termed as a reference IscB polypeptide. The properties of a new IscB polypeptide can be evaluated with a reference IscB polypeptide as a reference from which the new IscB polypeptide is derived.
  • an original IscB polypeptide e.g., a wild-type IscB polypeptide
  • the original IscB polypeptide is mutated to generate the new IscB polypeptide.
  • the original IscB polypeptide is a reference of the
  • one or more of the properties (e.g., endonuclease activity, nickase activity) of the new IscB polypeptide can be compared with the reference IscB polypeptide from which the new IscB polypeptide is derived.
  • nickase refers to two different kinds of nuclease. Both endonuclease and nickase can function on dsDNA. Endonuclease cleaves both strands of a dsDNA, leading to double-strand cleavage.nickase cleaves either the nontarget strand or the target strand of a dsDNA but not both, leading to single-strand cleavage (or “nick” ) on either the nontarget strand or the target strand but not both. As used herein, the term “nick” is used interchangeably with “ssDNA cleavage” .
  • endonuclease activity refers to a nuclease activity capable of cleaving both strands of a dsDNA, leading to double-strand cleavage.
  • Nickase activity refers to a nuclease activity capable of cleaving either the nontarget strand or the target strand of a dsDNA but not both, leading to single-strand cleavage (or “nick” ) on either the nontarget strand or the target strand but not both.
  • IscB endonuclease refers to an IscB polypeptide having endonuclease activity.
  • IscB nickase refers to an IscB polypeptide having nickase activity.
  • the term “dead IscB” refers to an IscB polypeptide (1) having low or no endonuclease activity; and (2) having low or no nickase activity.
  • the term “having (e.g., endonuclease, nickase) activity” means that the indicated activity is observable and detectable, for example, as detected according to the general principle of the experimental procedure in the art, such as described herein in the disclosure (e.g., the evaluation assay in Example 1) .
  • the IscB polypeptide having (e.g., endonuclease, nickase) activity is generated from a reference IscB polypeptide
  • such term “having (e.g., endonuclease, nickase) activity” may also be described as “substantially retaining (e.g., endonuclease, nickase) activity” of the reference IscB polypeptide, indicating that it has a great or significant part of the activity of the reference IscB polypeptide.
  • the term “having low (e.g., endonuclease, nickase) activity” means that the indicated activity is low, for example, as detected according to the general principle of the experimental procedure in the art, such as described herein in the disclosure (e.g., the evaluation assay in Example 1) .
  • the term “having no (e.g., endonuclease, nickase) activity” means that the indicated activity is absent, or not observable or detectable, for example, as detected according to the general principle of the experimental procedure in the art, such as described herein in the disclosure (e.g., the evaluation assay in Example 1) .
  • the IscB polypeptide having low or no (e.g., endonuclease, nickase) activity is generated from a reference IscB polypeptide
  • such term “having low or no (e.g., endonuclease, nickase) activity” may also be described as “substantially lacking (e.g., endonuclease, nickase) activity” of the reference IscB polypeptide, indicating that it has a great or significant reduction of (e.g., endonuclease, nickase) activity compared to the activity of the reference IscB polypeptide (e.g., a corresponding wild-type IscB) .
  • nickase activity of a (e.g., engineered) IscB polypeptide compared to the nickase activity of a reference IscB polypeptide, it does not mean to acknowledge that the reference IscB polypeptide is a nickase.
  • a reference IscB polypeptide that has endonuclease activity may also show positive results in a nickase activity evaluation assay due to its capability of cleaving one strand of a dsDNA, which however does not make it a nickase.
  • the disclosure provides various IscB polypeptides, including IscB endonucleases, IscB nickase, and dead IscB, suitable for various applications.
  • the disclosure provides an IscB polypeptide.
  • the IscB polypeptide is an engineered IscB polypeptide.
  • IscB identified by the applicant have endonuclease activity in eukaryotic cells, making them suitable for various applications in eukaryotic cells. They can also serve as engineering basis to develop various engineered IscB polypeptides according to the principle of the disclosure.
  • the disclosure provides an IscB polypeptide:
  • the IscB polypeptide is isolated.
  • the scaffold sequence of the cognate (corresponding) ⁇ RNAs of the IscB polypeptides of SEQ ID NOs: 26-33 are set forth in SEQ ID NOs: 92-99, respectively.
  • IscB that may also serve as basis for engineering according to the principle of the disclosure and their cognate (corresponding) ⁇ RNAs are disclosed in PCT/US2021/056361 and Altae-Tran, Han et al. “The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. ” Science (New York, N. Y. ) vol. 374, 6563 (2021) : 57-65. doi: 10.1126/science. abj6856 (and all its supplementary materials) , the entire contents of which, including any sequence listing and drawings, are incorporated herein by reference in their entireties.
  • the disclosure provides an IscB polypeptide:
  • the IscB polypeptide is isolated.
  • the scaffold sequence of the cognate (corresponding) ⁇ RNAs of the IscB polypeptides of SEQ ID NOs: 26-91 are set forth in SEQ ID NOs: 92-157, respectively.
  • the disclosure provide engineered IscB polypeptides developed from the IscB polypeptide of the disclosure, for example, by introducing one or more mutation into the IscB polypeptide of the disclosure.
  • the IscB polypeptides (e.g., the engineered IscB polypeptides) of the disclosure may be used in combination with a guide nucleic acid as described herein to constitute a system comprising the IscB polypeptide and the guide nucleic acid, i.e., an IscB system.
  • the disclosure provides a system comprising:
  • IscB polypeptide e.g., the engineered IscB polypeptide
  • fusion protein of the disclosure or a polynucleotide (e.g., a DNA, an RNA) encoding the IscB polypeptide or the fusion protein, and
  • a guide nucleic acid or a polynucleotide e.g., a DNA, an RNA
  • the guide nucleic acid comprising:
  • a guide sequence capable of hybridizing to a target sequence of a target DNA (e.g., a target dsDNA) , thereby guiding the complex to the target DNA.
  • a target DNA e.g., a target dsDNA
  • the system is a complex comprising the IscB polypeptide complexed with the guide nucleic acid.
  • the complex further comprises the target DNA hybridized with the guide sequence.
  • the guide nucleic acid is so designed to target the IscB polypeptide to the target DNA, by relying on the hybridization between the guide sequence and the target DNA.
  • the system is a composition comprising the component (1) and the component (2) .
  • the scaffold sequence is (e.g., immediately) 3’ to the guide sequence.
  • the guide nucleic acid is a guide RNA (gRNA) , e.g., a single guide RNA (sgRNA) .
  • gRNA guide RNA
  • sgRNA single guide RNA
  • the system further comprises a donor polynucleotide for integration or insertion into the target DNA.
  • the disclosure provides a guide nucleic acid described herein.
  • IscB having low or no endonuclease activity since the endonuclease activity of IscB may not be desired for such applications, for example, base editing and prime editing that may prefer to use a IscB nickase or a dead IscB.
  • one of the objects of the disclosure is to provide IscB having low or no endonuclease activity.
  • Another object of the disclosure is to provide IscB nickase.
  • Yet another object of the disclosure is to provide dead IscB.
  • IscB may have endonuclease activity to cleave both the target and nontarget strands of a dsDNA.
  • IscB may be engineered to be an IscB variant, which has reduced endonuclease activity to cleave both the strands compared to the original IscB without such engineering, but has nickase activity to cleave one of the target strand and the nontarget strand, and such a IscB variant is referred to as “IscB nickase” or “nIscB” herein.
  • this is achieved by mutagenesis at a conserved amino acid residue in a conservative motif of RuvC nuclease domain and/or HNH nuclease function of IscB.
  • IscB contains a RuvC nuclease domain separated into three segments: RuvC I, II, and III domains, which is responsible for single-strand cleavage at the nontarget strand of a dsDNA, and a HNH nuclease domain, which is responsible for single-strand cleavage at the target strand of a dsDNA, together leading to double-strand cleavage of the dsDNA.
  • the motif was composed of conserved amino acid residue H 1 (where “1” means that it is the first H in the motif) and conserved amino acid residue N 1 (where “1” means that it is the first N in the motif) and 12 to 17 undefined amino acid residues x between H 1 and N 1 (shown as “ ⁇ 12-17x ⁇ ” ) , followed by 8 undefined amino acid residues x (shown as “ ⁇ 8x ⁇ ” ) and further by one of three different conditions:
  • conserved amino acid residue it means that the amino acid residue is constant (not changed) as indicated across all the analyzed IscB.
  • conserve motif it means that the motif has a constant (not changed) pattern as indicated across all the analyzed IscB. See “CLUSTAL multiple sequence alignment by Kalign (3.3.1) ” in the “EXAMPLARY SEQUENCES” section of the disclosure.
  • HISCB016 (SEQ ID NO: 29) as an example, it contains motif P in RuvC I domain, motif INK in RuvC II domain, and motif HI in RuvC III domain, and motif VIPKHEHGSDTIA IVGLCSGC DLV in HNH domain (the conserved amino acids are double-underlined) .
  • KraIscB3 (SEQ ID NO: 35) as another example, it contains motif T in RuvC I domain, motif VGN in RuvC II domain, and motif TN in RuvC III domain, and motif IVPRSRGGGSRES LTLACRPC ER in HNH domain (the conserved amino acids are double-underlined) .
  • the endonuclease activity of the reference IscB polypeptide may be reduced, making the resulting engineered IscB polypeptides suitable for various applications that do not want endonuclease activity, and in particular, the resulting engineered IscB polypeptides may have nickase activity, making them particularly suitable for applications that need nickase activity, such as, base editing.
  • the endonuclease activity of the reference IscB polypeptide may be reduced, making the resulting engineered IscB polypeptides suitable for various applications that do not want endonuclease activity, and in particular, the resulting engineered IscB polypeptides may also have nickase activity, making them particularly suitable for applications that need nickase activity, such as, base editing.
  • an amino acid mutation e.g., deletion
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid substitution at: (1) D and/or G of motif DxG in the RuvC I domain of a reference IscB polypeptide; (2) E and/or F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide; and/or (3) H, D, and/or A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide; wherein x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid substitution at: (1) D and/or G of motif DxG in the RuvC I domain of the reference IscB polypeptide; (2) E and/or F of motif ExxxF in the RuvC II domain of the reference IscB polypeptide; and/or (3) H, D, and/or A of motif HxxDA in the RuvC III domain of the reference IscB polypeptide; thereby generating the engineered IscB polypeptide; wherein x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid deletion at one or more (or all) amino acid residues of the RuvC I domain (e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain) , the RuvC II domain (e.g., one or more (or all) amino acid residues of the motif ExxxF in the RuvC I domain) , and/or the RuvC III domain (e.g., one or more (or all) amino acid residues of the motif HxxDA in the RuvC I domain) of a reference IscB polypeptide; wherein x is any amino acid residue.
  • the RuvC I domain e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain
  • the RuvC II domain e.g., one or more (or all) amino acid residues of the motif ExxxF in the RuvC I domain
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid deletion at one or more (or all) amino acid residues of the RuvC I domain (e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain) , the RuvC II domain (e.g., one or more (or all) amino acid residues of the motif ExxxF in the RuvC I domain) , and/or the RuvC III domain (e.g., one or more (or all) amino acid residues of the motif HxxDA in the RuvC I domain) of the reference IscB polypeptide, thereby generating the engineered IscB polypeptide; wherein x is any amino acid residue.
  • the RuvC I domain e.g., one or more (or all) amino acid residues of the motif DxG in the RuvC I domain
  • the RuvC II domain e
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid substitution at H 1 , N 1 , H 2 , H 3 , N 2 and/or K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide; wherein x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid substitution at H 1 , N 1 , H 2 , H 3 , N 2 and/or K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of the reference IscB polypeptide, thereby generating the engineered IscB polypeptide; wherein x is any amino acid residue.
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides an engineered IscB polypeptide comprising an amino acid deletion at one or more (or all) amino acid residues of the HNH domain (e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, wherein x is any amino acid residue) of a reference IscB polypeptide.
  • the HNH domain e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, wherein x is any amino acid residue
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide an amino acid deletion at one or more (or all) amino acid residues of the HNH domain (e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, wherein x is any amino acid residue) of the reference IscB polypeptide, thereby generating the engineered IscB polypeptide.
  • an amino acid deletion at one or more (or all) amino acid residues of the HNH domain (e.g., one or more (or all) amino acid residues of the motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, where
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide has nickase activity.
  • an engineered IscB polypeptide comprising:
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide (1) has low or no endonuclease activity and (2) has low or no nickase
  • the disclosure provides a method of generating an engineered IscB polypeptide, comprising introducing into a reference IscB polypeptide:
  • the engineered IscB polypeptide has reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide or has no endonuclease activity.
  • the engineered IscB polypeptide (1) has low or no endonucle
  • the resulting engineered IscB polypeptide generated from a refence IscB polypeptide substantially lacks endonuclease activity so as to be able to be used for applications that do not want endonuclease activity.
  • the engineered IscB polypeptide has low or no endonuclease activity, e.g., substantially lacking endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide, e.g., having reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide.
  • the engineered IscB polypeptide has nickase activity, e.g., substantially retaining nickase activity of the reference IscB polypeptide, e.g., having at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more of the nickase activity of the reference IscB polypeptide.
  • the engineered IscB polypeptide has nickase activity against the target strand of a target dsDNA.
  • the engineered IscB polypeptide has nickase activity against the non-target strand of a target dsDNA.
  • the engineered IscB polypeptide has low or no nickase activity, e.g., substantially lacking nickase activity compared with the nickase activity of the reference IscB polypeptide, e.g., having reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) nickase activity compared with the nickase activity of the reference IscB polypeptide.
  • the engineered IscB polypeptide has (1) low or no endonuclease activity, e.g., substantially lacking endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide, e.g., having reduced (e.g., a reduction by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%or by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) endonuclease activity compared with the endonuclease activity of the reference IscB polypeptide; and
  • amino acid substitution at one or more of the conservative motifs, especially at one or more of the conserved amino acids, of a reference IscB polypeptide may be a conservative amino acid substitution or a non-conservative amino acid substitution.
  • the amino acid substitution comprises or is a non-conservative amino acid substitution.
  • the amino acid substitution at D of motif DxG in the RuvC I domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at G of motif DxG in the RuvC I domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at H of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at D of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the amino acid substitution at K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a non-conservative amino acid substitution.
  • the non-conservative amino acid substitution is a non-conservative amino acid substitution with A.
  • the amino acid substitution comprises or is a conservative amino acid substitution.
  • the amino acid substitution at D of motif DxG in the RuvC I domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at G of motif DxG in the RuvC I domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at H of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at D of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the amino acid substitution at K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is a conservative amino acid substitution.
  • the conservative amino acid substitution is a non-conservative amino acid substitution with A.
  • the amino acid substitution comprises or is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid; and optionally, with a non-polar amino acid, such as, A.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at D of motif DxG in the RuvC I domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at G of motif DxG in the RuvC I domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at F of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at H of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at D of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at A of motif HxxDA in the RuvC III domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the amino acid substitution at K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is an amino acid substitution with a non-polar amino acid, a polar amino acid, a positively charged amino acid, or a negatively charged amino acid.
  • the non-polar amino acid is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; and optionally the non-polar amino acid comprises or is Alanine (Ala/A) .
  • the polar amino acid is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; and optionally the positively charged amino acid comprises or is Arginine (Arg/R) .
  • the negatively charged amino acid is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the amino acid substitution is an amino acid substitution with Alanine (Ala/A) or Arginine (Arg/R) .
  • the reference IscB polypeptide based on which the engineered IscB polypeptide of the disclosure may be designed and engineered may be any IscB that share the same RuvC nuclease domain and HNH nuclease domain for engineering as the IscB listed in the disclosure.
  • the reference IscB polypeptide may be a wild-type IscB polypeptide (e.g., OgeuIscB of SEQ ID NO: 89) or also an engineered IscB polypeptide (e.g., enOgeuIscB of SEQ ID NO: 158) developed from a wild-type IscB polypeptide (e.g., OgeuIscB of SEQ ID NO: 89) .
  • the engineered IscB polypeptide of the disclosure may retain one or more (e.g., substantially all or all) of the amino acid changes in the sequence of the reference engineered IscB polypeptide (e.g., enOgeuIscB of SEQ ID NO: 158) compared to the sequence of a corresponding wild-type IscB polypeptide (e.g., OgeuIscB of SEQ ID NO: 89) from which the reference engineered IscB polypeptide (e.g., enOgeuIscB of SEQ ID NO: 158) is developed.
  • a corresponding wild-type IscB polypeptide e.g., OgeuIscB of SEQ ID NO: 89
  • the engineered IscB polypeptide retains one or more (e.g., substantially all or all) of the amino acid changes in the sequence of SEQ ID NO: 158 compared to the sequence of SEQ ID NO: 89.
  • the reference IscB polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 26-91 and 158, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 26-91 and 158.
  • the scaffold sequence of the cognate (corresponding) ⁇ RNAs of the IscB polypeptides of SEQ ID NOs: 26-91 and 158 are set forth in SEQ ID NOs: 92-157 and 159, respectively.
  • the reference IscB polypeptide of the disclosure is CRISPR-associated.
  • the disclosure provides a map and a guide to identify the conservative motif and conserved amino acid residues therein of unlisted IscB known in the art or not.
  • FIG. 8 shows the domain organization of IscB. P1D, P1 interaction domain; TID, TAM-interaction domain. RuvC domain is separated into three segments: RuvC I, II, and III.
  • FIG. 9 shows the predicted domain origination of HISCB016 and the amino acid residue at the boundary between two domains. Each amino acid residue at the boundary is assigned to the domain on its N’ terminal. For example, the amino acid residue at position 86 is assigned to RuvC I domain instead of Bridge Helix.
  • FIG. 10 shows the predicted domain origination of HISCB017 and the amino acid residue at the boundary between two domains. Each amino acid residue at the boundary is assigned to the domain on its N’ terminal. For example, the amino acid residue at position 85 is assigned to RuvC I domain instead of Bridge Helix.
  • the rough positions of the RuvC I, II, and III domains and the HNH domain of an unlisted IscB in its amino acid sequence can be determined by sequence alignment with, for example, HISCB016 (SEQ ID NO: 29) .
  • HISCB008 SEQ ID NO: 26
  • the conservative motifs DxG, ExxxF, HxxDA, and H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ of HISCB008 can be directly identified by sequence alignment with HISCB016 without firstly identifying the RuvC I, II, and III domains and the HNH domain of HISCB008.
  • the D of motif DxG in the RuvC I domain is at a position of the reference IscB polypeptide corresponding to position D61 of SEQ ID NO: 29.
  • amino acid residue x in polypeptide A is aligned with amino acid residue y in polypeptide B, it is said that amino acid residue x in polypeptide A is corresponding to amino acid residue y in polypeptide B.
  • D62 in HISCB008 is aligned with D61 in HISCB016, it is thus said that D62 in HISCB008 is corresponding to D61 in HISCB016.
  • This provides a way to describe an amino acid residue in a polypeptide of which the position of the amino acid residue has not been numbered or cannot be numbered yet.
  • This embodiment means that the D of motif DxG in the RuvC I domain in a reference IscB polypeptide is identified by looking for a D of motif DxG in the RuvC I domain in the reference IscB polypeptide that is aligned with (corresponding to) the D at position D61 of SEQ ID NO: 29 (which is HISCB016) , no matter what the specific position of the D in the reference IscB polypeptide is.
  • the G of motif DxG in the RuvC I domain is at a position of the reference IscB polypeptide corresponding to position G63 of SEQ ID NO: 29.
  • the E of motif ExxxF in the RuvC II domain is at a position of the reference IscB polypeptide corresponding to position E193 of SEQ ID NO: 29.
  • the F of motif ExxxF in the RuvC II domain is at a position of the reference IscB polypeptide corresponding to position F197 of SEQ ID NO: 29.
  • the H of motif HxxDA in the RuvC III domain is at a position of the reference IscB polypeptide corresponding to position H340 of SEQ ID NO: 29.
  • the D of motif HxxDA in the RuvC III domain is at a position of the reference IscB polypeptide corresponding to position D343 of SEQ ID NO: 29.
  • the A of motif HxxDA in the RuvC III domain is at a position of the reference IscB polypeptide corresponding to position A344 of SEQ ID NO: 29.
  • the H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position H248 of SEQ ID NO: 29.
  • the N 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position N262 of SEQ ID NO: 29.
  • the H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position H271 of SEQ ID NO: 29.
  • the H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position H275 of SEQ ID NO: 29.
  • the N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position N236 of SEQ ID NO: 35.
  • the K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain is at a position of the reference IscB polypeptide corresponding to position K239 of SEQ ID NO: 35.
  • the engineered IscB polypeptide has a sequence identity of at least about 50% (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%) to the amino acid sequence of the reference IscB polypeptide.
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the engineered IscB polypeptide comprises (1) an amino acid substitution of D61A, D61E, D61Q, D61R, G63A, G63E, G63Q, G63R, E193A, E193D, E193Q, E193R, F197A, F197E, F197Q, F197R, H340A, H340E, H340Q, H340R, D343A, D343E, D343Q, D343R, A344G, A344E, A344Q, and/or A344R.
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the engineered IscB polypeptide comprises (2) an amino acid substitution of H248A, H248R, H271A, H271R, H275A, and/or H275R. In some embodiments, the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 29, and the engineered IscB polypeptide comprises both (1) and (2) .
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the engineered IscB polypeptide comprises an amino acid substitution of D61A.
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and the engineered IscB polypeptide comprises an amino acid substitution of D61A and H248A.
  • the engineered IscB polypeptide comprises SEQ ID NO: 170 or 171 or a N-terminal truncation of SEQ ID NO: 170 or 171 without N-terminal Methionine (M) .
  • a protein is encoded by a coding sequence with 5’ start codon ATG (encoding Methionine (M) ) to initiate the transcription of the coding sequence, and thus the protein carries a corresponding Methionine (M) at its N-terminal end.
  • N-terminal Methionine (M) may not be needed in some cases, for example when such a protein is fused at the C-terminal of another protein, and in such a case, the N-terminal Methionine (M) of the protein may be removed /truncated, leaving a N-terminal truncation of the protein without N-terminal Methionine (M) , and correspondingly the start codon ATG of the coding sequence encoding the protein may be removed.
  • N-terminal truncation of SEQ ID NO: 170 or 171 without N-terminal Methionine (M) it means that the Methionine (M) (corresponding to the start codon ATG) at the N-terminal end of SEQ ID NO: 170 or 171 in length of 513 amino acids is truncated, leaving N-terminal truncation of SEQ ID NO: 170 or 171 in length of 512 amino acids lacking the N-terminal Methionine (M) .
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and the engineered IscB polypeptide comprises (1) an amino acid substitution of D60A, D60E, D60Q, D60R, G62A, G62E, G62Q, G62R, E190A, E190D, E190Q, E190R, F194A, F194E, F194Q, F194R, H333A, H333E, H333Q, H333R, D336A, D336E, D336Q, D336R, A337G, A337E, A337Q, and/or A337R.
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and the engineered IscB polypeptide comprises (2) an amino acid substitution of H244A, H244R, H267A, H267R, H271A, and/or H271R. In some embodiments, the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and the engineered IscB polypeptide comprises both (1) and (2) .
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 89 or 158
  • the engineered IscB polypeptide comprises (1) an amino acid substitution of D61A, D61E, D61Q, D61R, G63A, G63E, G63Q, G63R, E193A, E193D, E193Q, E193R, F197A, F197E, F197Q, F197R, H340A, H340E, H340Q, H340R, D343A, D343E, D343Q, D343R, A344G, A344E, A344Q, and/or A344R.
  • the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 89 or 158, and the engineered IscB polypeptide comprises (2) an amino acid substitution of H247A. In some embodiments, the reference IscB polypeptide comprises the amino acid sequence of SEQ ID NO: 89 or 158, and the engineered IscB polypeptide comprises both (1) and (2) .
  • the engineered IscB polypeptide comprises any one of SEQ ID NOs: 164-166 or a N-terminal truncation of any one of SEQ ID NOs: 164-166 without N-terminal Methionine (M) .
  • a protein is encoded by a coding sequence with 5’ start codon ATG (encoding Methionine (M) ) to initiate the transcription of the coding sequence, and thus the protein carries a corresponding Methionine (M) at its N-terminal end.
  • N-terminal Methionine (M) may not be needed in some cases, for example when such a protein is fused at the C-terminal of another protein, and in such a case, the N-terminal Methionine (M) of the protein may be removed /truncated, leaving a N-terminal truncation of the protein without N-terminal Methionine (M) , and correspondingly the start codon ATG of the coding sequence encoding the protein may be removed.
  • N-terminal truncation of any one of SEQ ID NOs: 164-166 without N-terminal Methionine (M) it means that the Methionine (M) (corresponding to the start codon ATG) at the N-terminal end of any one of SEQ ID NOs: 164-166 in length of 496 amino acids is truncated, leaving N-terminal truncation of any one of SEQ ID NOs: 164-166 in length of 495 amino acids lacking the N-terminal Methionine (M) .
  • the engineered IscB polypeptide does not comprises an amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises a non-conservative amino acid substitution at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises an amino acid substitution of E with a non-polar amino acid residue at E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises an amino acid substitution E-to-Aat E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises an E-to-Aamino acid substitution at the E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide of SEQ ID NO: 52.
  • the engineered IscB polypeptide does not comprises an amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises a non-conservative amino acid substitution at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • a reference IscB polypeptide e.g., SEQ ID NO: 52
  • the engineered IscB polypeptide does not comprises an amino acid substitution of H with a non-polar amino acid residue at H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises an amino acid substitution H-to-Aat H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide (e.g., SEQ ID NO: 52) .
  • the engineered IscB polypeptide does not comprises an H-to-Aamino acid substitution at the H 1 in motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide of SEQ ID NO: 52.
  • the reference IscB polypeptide is not or does not comprise the amino acid sequence of SEQ ID NO: 52.
  • the E of motif ExxxF in the RuvC II domain of a reference IscB polypeptide is not E157 of SEQ ID NO: 52.
  • the H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of a reference IscB polypeptide is not H212 of SEQ ID NO: 52.
  • the engineered IscB polypeptide does not comprises E157A in SEQ ID NO: 52.
  • the engineered IscB polypeptide does not comprises H212A in SEQ ID NO: 52.
  • the engineered IscB polypeptide does not comprises E157A and H212A in SEQ ID NO: 52.
  • the engineered IscB polypeptide is not any one of AwaIscB-E157A, AwaIscB-H212A, or AwaIscB-E157A+H212A.
  • the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure is capable of recognizing a target adjacent motif (TAM) immediately 3' to a protospacer sequence on a nontarget strand of a dsDNA, and wherein the TAM is 5’-NNNNNN-3’, wherein N is A, T, G, or C.
  • TAM target adjacent motif
  • the TAM is 5’-NNNGAN-3’, wherein N is A, T, G, or C.
  • the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure further comprises a functional domain.
  • the functional domain is fused to the IscB polypeptide with or without a linker to form a fusion protein.
  • the disclosure provides a fusion protein comprising the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure and a functional domain.
  • the linker is a GS linker containing multiple glycine (GS) and serine (S) residues, XTEN linker, XTEN&GS linker containing XTEN linker, or bpSV40 NLS&GS linker containing bpSV40 NLS.
  • the IscB polypeptide further comprises the functional domain fused at the N-terminal, C-terminal, or both terminals of the IscB polypeptide, or fused internally with respect to the IscB polypeptide, to form a fusion protein.
  • the functional domain is selected from the group consisting of an exonuclease, a reverse transcriptase (such as, Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) ) , a nuclear localization signal (NLS) , a nuclear export signal (NES) , a base editing domain, for example, a deaminase or a catalytic domain thereof, a base excising domain, an uracil glycosylase inhibitor (UGI) or a catalytic domain thereof, a glycosylase or a catalytic domain thereof, for example, an uracil glycosylase (UNG) or a catalytic domain thereof, a methylpurine glycosylase (MPG) or a catalytic domain thereof, a methylase or a catalytic domain thereof, a demethylase or a catalytic domain thereof, an transcription activating domain (e.g., VP64 or VPR)
  • coli dihydrofolate reductase ecDHFR
  • a histone residue modification domain e.g., a nuclease catalytic domain (e.g., FokI)
  • a transcription modification factor e.g., a light gating factor, a chemical inducible factor, a chromatin visualization factor
  • a targeting polypeptide for providing binding to a cell surface portion on a target cell or a target cell type a reporter (e.g., fluorescent) polypeptide or a detection label (e.g., GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP)
  • a localization signal e.g., a polypeptide targeting moiety, a DNA binding domain (e.g., MBP, Lex A DBD, Gal4 DBD) , an epitope tag (e.g., His, myc, V5, FLAG, HA, VSV-G, Trx, etc
  • the NLS is SV40 NLS, bpSV40 NLS (BP NLS; bpNLS, or NP NLS (Xenopus laevis Nucleoplasmin NLS; nucleoplasmin NLS) .
  • the exonuclease is T5 exonuclease (T5E) .
  • the deaminase or catalytic domain thereof is an adenine deaminase or a catalytic domain thereof (e.g., tRNA adenosine deaminase (TadA) , such as, TadA8e, TadA8.17, TadA8.20, TadA9, TadA8e-V106W, TadA8E V106W+D108Q TadA-CDa, TadA-CDb, TadA-CDc, TadA-CDd, TadA-CDe, TadA-dual, T AD AC-1.2, T AD AC-1.14, T AD AC-1.17, T AD AC-1.19, T AD AC-2.5, T AD AC-2.6, T AD AC-2.9, T AD AC-2.19, T AD AC-2.23, TadA8e-N46L, TadA8e-N46P) .
  • TadA tRNA adenosine deaminase
  • the deaminase or catalytic domain thereof is a cytosine deaminase or a catalytic domain thereof (e.g., an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, an activation induced deaminase (AID) , a cytidine deaminase 1 from Petromyzon marinus (pmCDA1) , or a functional variant thereof, e.g., APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, hAPOBEC3-W104A) .
  • APOBEC1 apolipoprotein B mRNA-editing complex
  • AID activation induced deaminase
  • AID activation induced deaminase
  • AID activation induced
  • the UGI is human UGI domain.
  • the fusion protein comprises the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure and a deaminase domain.
  • IscB polypeptide e.g., the engineered IscB polypeptide
  • deaminase domain e.g., the adenine deaminases.
  • adenine deaminases are known in the art, including, for example, those listed in WO2020/181195, which is incorporated herein by reference in its entirety.
  • the deaminase domain is a deaminase domain substantially capable of deaminating adenine, guanine, hypoxanthine, cytidine, thymine, and/or uracil.
  • the fusion protein comprises the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure and an adenine deaminase domain, with or without a linker between the IscB polypeptide and the adenine deaminase domain.
  • the fusion protein comprises, from N-terminal to C-terminal, the IscB polypeptide and an adenine deaminase domain, with or without a linker between the IscB polypeptide and the adenine deaminase domain.
  • the IscB polypeptide comprises, from N-terminal to C-terminal, an adenine deaminase domain, the IscB polypeptide, and an adenine deaminase domain, individually with or without a linker between any two adjacent components of the fusion protein.
  • the adenine deaminase domain is tRNA adenosine deaminase (TadA) or a functional variant thereof, such as, TadA8e, TadA8.17, TadA8.20, TadA9, TadA8e-V106W, TadA8E V106W+D108Q TadA-CDa, TadA-CDb, TadA-CDc, TadA-CDd, TadA-CDe, TadA-dual, T AD AC-1.2, T AD AC-1.14, T AD AC-1.17, T AD AC-1.19, T AD AC-2.5, T AD AC-2.6, T AD AC-2.9, T AD AC-2.19, T AD AC-2.23, TadA8e-N46L, TadA8e-N46P.
  • TadA tRNA adenosine deaminase
  • the fusion protein comprises the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure and a cytosine deaminase domain.
  • the fusion protein comprises the IscB polypeptide, a cytosine deaminase domain, and a UGI domain. In some embodiments, the fusion protein comprises the IscB polypeptide, a cytosine deaminase domain, and two UGI domains.
  • the fusion protein comprises, from N-terminal to C-terminal, a cytosine deaminase domain, the IscB polypeptide, a UGI domain, and a UGI domain, individually with or without a linker between any two adjacent components of the fusion protein.
  • the cytosine deaminase domain is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, an activation induced deaminase (AID) , a cytidine deaminase 1 from Petromyzon marinus (pmCDA1) , or a functional variant thereof, e.g., APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, hAPOBEC3-W104A.
  • APOBEC apolipoprotein B mRNA-editing complex
  • AID activation induced deaminase
  • APOBEC1 a functional variant thereof, e.g., APOBEC1, APOBEC2,
  • the scaffold sequence is compatible with the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure and is capable of complexing with the IscB polypeptide.
  • the scaffold sequence may be a naturally occurring scaffold sequence identified along with the IscB polypeptide (e.g., OgeuIscB scaffold sequence of SEQ ID NO: 155) , or a variant thereof maintaining the ability to complex with the IscB polypeptide (e.g., enOgeuIscB scaffold sequence of SEQ ID NO: 159) .
  • the ability to complex with the IscB polypeptide is maintained as long as the secondary structure of the scaffold variant is substantially identical to the secondary structure of the corresponding, naturally occurring scaffold sequence.
  • a nucleotide deletion, insertion, or substitution in the primary sequence of the scaffold sequence may not necessarily change the secondary structure of the scaffold sequence (e.g., the relative locations and/or sizes of the stems, bulges, and loops of the scaffold sequence do not significantly deviate from that of the original stems, bulges, and loops) .
  • the nucleotide deletion, insertion, or substitution may be in a bulge or loop region of the scaffold sequence so that the overall symmetry of the bulge and hence the secondary structure remains largely the same.
  • the nucleotide deletion, insertion, or substitution may also be in the stems of the scaffold sequence so that the lengths of the stems do not significantly deviate from that of the original stems (e.g., adding or deleting one base pair in each of two stems correspond to 4 total base changes) .
  • Engineering of the scaffold sequence may be applied to improve the activity of the IscB system of the disclosure.
  • the scaffold sequence has substantially the same secondary structure as the secondary structure of any one of SEQ ID NOs: 92-157.
  • the scaffold sequence comprises a polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%) to the polynucleotide sequence of any one of SEQ ID NOs: 92-157.
  • the IscB polypeptide e.g., the engineered IscB polypeptide, the reference IscB polypeptide
  • the IscB polypeptide of the disclosure is used in combination with its cognate (corresponding) scaffold sequence.
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 26, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 92, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 27, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 93, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 28, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 94, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 29, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 95, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 30, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 96, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 31, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 97, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 32, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 98, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 33, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 99, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 34, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 100, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 35, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 101, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 36, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 102, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 37, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 103, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 38, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 104, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 39, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 105, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 40, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 106, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 41, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 107, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 42, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 108, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 43, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 109, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 44, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 110, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 45, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 111, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 46, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 112, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 47, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 113, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 48, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 114, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 49, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 115, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 50, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 116, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 51, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 117, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 52, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 118, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 53, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 119, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 54, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 120, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 55, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 121, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 56, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 122, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 57, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 123, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 58, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 124, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 59, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 125, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 60, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 126, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 61, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 127, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 62, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 128, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 63, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 129, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 64, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 130, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 65, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 131, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 66, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 132, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 67, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 133, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 68, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 134, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 69, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 135, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 70, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 136, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 71, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 137, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 72, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 138, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 73, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 139, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 74, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 140, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 75, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 141, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 76, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 142, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 77, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 143, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 78, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 144, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 79, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 145, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 80, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 146, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 81, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 147, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 82, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 148, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 83, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 149, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 84, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 150, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 85, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 151, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 86, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 152, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 87, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 153, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 88, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 154, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 89, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 155, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 90, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 156, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
  • the IscB polypeptide (e.g., the IscB polypeptide, the engineered IscB polypeptide, the reference IscB polypeptide) of the disclosure comprises the amino acid sequence of SEQ ID NO: 91, or an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ ID NO: 91, and the scaffold sequence comprises the polynucleotide sequence of SEQ ID NO: 157, or an polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%
  • the protospacer sequence comprises about or at least about 14 contiguous nucleotides of the target DNA, e.g., about or at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more contiguous nucleotides of the target DNA, or in a numerical range between any two of the preceding values, e.g., from about 14 to about 50, or from about 17 to about 22 contiguous nucleotides of the target DNA.
  • the protospacer sequence comprises about 16 contiguous nucleotides of the target DNA.
  • the protospacer sequence comprises about 16 contiguous nucleotides of the target DNA.
  • the protospacer sequence is immediately 5’ to a target adjacent motif (TAM) .
  • TAM target adjacent motif
  • the TAM is 5’-NNNNNN-3’, wherein N is A, T, G, or C.
  • the TAM is 5’-NNNGAN-3’, wherein N is A, T, G, or C.
  • the target sequence comprises about or at least about 14 contiguous nucleotides of the target DNA, e.g., about or at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more contiguous nucleotides of the target DNA, or in a numerical range between any two of the preceding values, e.g., from about 14 to about 50, or from about 17 to about 22 contiguous nucleotides of the target DNA.
  • the target sequence comprises about 16 contiguous nucleotides of the target DNA.
  • the target sequence is on the target
  • the reverse complementary sequence of the target sequence is immediately 5’ to a target adjacent motif (TAM) .
  • TAM target adjacent motif
  • the TAM is 5’-NNNNNN-3’, wherein N is A, T, G, or C.
  • the TAM is 5’-NNNGAN-3’, wherein N is A, T, G, or C.
  • the guide sequence is about or at least about 14 nucleotides in length, e.g., about or at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more nucleotides in length, or in a length of a numerical range between any two of the preceding values, e.g., in a length of from about 14 to about 50 nucleotides, or from about 17 to about 22 nucleotides.
  • the guide sequence is about 16 nucleotides in length.
  • the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (fully) reverse complementary to the target sequence; (2) the guide sequence contains no more than 5, 4, 3, 2, or 1 mismatch or contains no mismatch with the target sequence; or (3) the guide sequence comprises no mismatch with the target sequence in the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides at the 3’ end of the guide sequence. In some embodiments, (1) the guide sequence is about 100% (fully) reverse complementary to the target sequence.
  • the system comprises two or more guide nuclei acids comprising two or more guide sequences capable of hybridizing to two or more target sequences of the same target DNA or different target DNAs, wherein the two or more guide sequences are the same or different, and wherein the two or more target sequences are the same or different.
  • the IscB polypeptide (e.g., the engineered IscB polypeptide) of the disclosure is capable of targeting a target DNA (e.g., a target dsDNA) .
  • the target DNA is a target dsDNA.
  • the DNA is a eukaryotic DNA.
  • the DNA is a DNA in a eukaryotic cell.
  • the DNA is a gene, e.g., a human gene.
  • the dsDNA comprises a protospacer sequence on a nontarget strand of the dsDNA, wherein the dsDNA comprises a target deoxyribonucleotide (e.g., dA, dT, dC, dG) at a position of the protospacer sequence selected from the group consisting of position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, and a combination thereof; or wherein the target deoxyribonucleotide is at a position of the protospacer sequence between position 2 and position 12 or between position 3 and position 14, both inclusive.
  • a target deoxyribonucleotide e.g., dA, dT, dC, dG
  • the target deoxyribonucleotide is the N 2 nucleotide in a motif of N 1 N 2 N 3 , wherein N 1 , N 2 , or N 3 is A, T, G, or C.
  • the disclosure provides a polynucleotide encoding the IscB polypeptide (e.g., the engineered IscB polypeptide) or the fusion protein of the disclosure.
  • the polynucleotide encodes a guide nucleic acid as described herein.
  • the polynucleotide encoding the guide nucleic acid is a DNA, a RNA, or a DNA/RNA mixture.
  • DNA/RNA mixture it refers to a nucleic acid comprising both one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • DNA or RNA it may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether consecutive or not, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • the guide nucleic acid is operably linked to or under the regulation of a promoter.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • Suitable promoters include, for example, a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇
  • the polynucleotide encoding the IscB polypeptide is a DNA, a RNA, or a DNA/RNA mixture.
  • DNA/RNA mixture it refers to a nucleic acid comprising both one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • DNA or RNA it may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether consecutive or not, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.
  • the polynucleotide encoding the IscB polypeptide is operably linked to or under the regulation of a promoter.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • Suitable promoters include, for example, a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇
  • the disclosure provides a delivery system comprising (1) the IscB polypeptide of the disclosure, the polynucleotide of the disclosure, or the system of the disclosure; and (2) a delivery vehicle.
  • the disclosure provides a vector comprising the polynucleotide of the disclosure.
  • the vector encodes a guide nucleic acid as described herein.
  • the vector is a plasmid vector, a recombinant AAV (rAAV) vector (vector genome) , or a recombinant lentivirus vector.
  • the disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the disclosure.
  • a simple introduction of AAV for delivery may refer to “Adeno-associated Virus (AAV) Guide” (addgene. org/guides/aav/) .
  • Adeno-associated virus when engineered to delivery, e.g., a protein-encoding sequence of interest, may be termed as a (r) AAV vector, a (r) AAV vector particle, or a (r) AAV particle, where “r” stands for “recombinant” .
  • the genome packaged in AAV vectors for delivery may be termed as a (r) AAV vector genome, vector genome, or vg for short, while viral genome may refer to the original viral genome of natural AAVs.
  • the serotypes of the capsids of rAAV particles can be matched to the types of target cells.
  • Table 2 of WO2018002719A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference) .
  • the rAAV particle comprising a capsid with a serotype suitable for delivery into ear cells (e.g., inner hair cells) .
  • the rAAV particle comprising a capsid with a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV. PHP.
  • the serotype of the capsid is AAV9 or a functional variant thereof.
  • rAAV particles may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650) .
  • the vector titers are usually expressed as vector genomes per ml (vg/ml) .
  • the vector titer is above 1 ⁇ 10 9 , above 5 ⁇ 10 10 , above 1 ⁇ 10 11 , above 5 ⁇ 10 11 , above 1 ⁇ 10 12 , above 5 ⁇ 10 12 , or above 1 ⁇ 10 13 vg/ml.
  • RNA sequence as a vector genome into a rAAV particle
  • systems and methods of packaging an RNA sequence as a vector genome into a rAAV particle is recently developed and applicable herein. See PCT/CN2022/075366, which is incorporated herein by reference in its entirety.
  • sequence elements described herein for DNA vector genomes when present in RNA vector genomes, should generally be considered to be applicable for the RNA vector genomes except that the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and/or the element in the DNA sequence is replaced with the corresponding element with a corresponding function in the RNA sequence or omitted because its function is unnecessary in the RNA sequence and/or an additional element necessary for the RNA vector genome is introduced.
  • dT is equivalent to U
  • dA is equivalent to A
  • a coding sequence e.g., as a sequence element of rAAV vector genomes herein, is construed, understood, and considered as covering and covers both a DNA coding sequence and an RNA coding sequence.
  • an RNA sequence can be transcribed from the DNA coding sequence, and optionally further a protein can be translated from the transcribed RNA sequence as necessary.
  • the RNA coding sequence per se can be a functional RNA sequence for use, or an RNA sequence can be produced from the RNA coding sequence, e.g., by RNA processing, or a protein can be translated from the RNA coding sequence.
  • a IscB polypeptide coding sequence encoding a IscB polypeptide covers either a IscB polypeptide DNA coding sequence from which a IscB polypeptide is expressed (indirectly via transcription and translation) or a IscB polypeptide RNA coding sequence from which a IscB polypeptide is translated (directly) .
  • a gRNA coding sequence encoding a gRNA covers either a gRNA DNA coding sequence from which a gRNA is transcribed or a gRNA RNA coding sequence (1) which per se is the functional gRNA for use, or (2) from which a gRNA is produced, e.g., by RNA processing.
  • 5’-ITR and/or 3’-ITR as DNA packaging signals may be unnecessary and can be omitted at least partly, while RNA packaging signals can be introduced.
  • a promoter to drive transcription of DNA sequences may be unnecessary and can be omitted at least partly.
  • a sequence encoding a polyA signal may be unnecessary and can be omitted at least partly, while a polyA tail can be introduced.
  • DNA elements of rAAV DNA vector genomes can be either omitted or replaced with corresponding RNA elements and/or additional RNA elements can be introduced, in order to adapt to the strategy of delivering an RNA vector genome by rAAV particles.
  • the disclosure provides a ribonucleoprotein (RNP) comprising the IscB polypeptide of the disclosure and a guide nucleic acid.
  • RNP ribonucleoprotein
  • the guide nucleic acid is as described herein.
  • the disclosure provides a lipid nanoparticle (LNP) comprising an RNA (e.g., mRNA) encoding the IscB polypeptide of the disclosure and a guide nucleic acid.
  • LNP lipid nanoparticle
  • the guide nucleic acid is as described herein.
  • the disclosure provides a cell comprising the IscB polypeptide of the disclosure, the system of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the rAAV particle of the disclosure, the RNP of the disclosure, or the LNP of the disclosure.
  • the system of the disclosure comprising the IscB polypeptide (e.g., the engineered IscB polypeptide) or the fusion protein of the disclosure has a wide variety of utilities, including modifying (e.g., cleaving, editing, deleting, inserting, translocating, inactivating, or activating) a target DNA in a multiplicity of cell types.
  • the systems have a broad spectrum of applications requiring high cleavage activity and/or small sizes, e.g., drug screening, disease diagnosis and prognosis, and treating various genetic disorders.
  • the system of the disclosure can be used to modify a target DNA, for example, to modify the translation and/or transcription of one or more genes of the cells.
  • the modification may lead to increased transcription /translation /expression of a gene.
  • the modification may lead to decreased transcription /translation /expression of a gene.
  • the disclosure provides a method of modifying a target DNA, comprising contacting the target DNA with the system of the disclosure, the vector of the disclosure, the ribonucleoprotein of the disclosure, or the lipid nanoparticle of the disclosure, wherein the guide sequence is capable of hybridizing to a target sequence of the target DNA, wherein the target DNA is modified by the system.
  • the modification includes indel event, dsDNA cleavage (double stranded break, DSB) , ssDNA cleavage (nicking) (e.g., on either target strand or nontarget stand of a dsDNA) , base editing (e.g., single base editing) , prime editing, repairing, and integration or insertion of exogenous donor (e.g., by homologous recombination) .
  • the target DNA is in a cell.
  • the cell is a eukaryotic cell (e.g., an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., mouse or rat) cell, a human cell, a plant cell, or a yeast cell) or a prokaryotic cell (e.g., a bacteria cell) .
  • a eukaryotic cell e.g., an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., mouse or rat) cell, a human cell, a plant cell, or a yeast cell
  • a prokaryotic cell e.g., a bacteria cell
  • the cell is from a plant or an animal.
  • the plant is a dicotyledon.
  • the dicotyledon is selected from the group consisting of soybean, cabbage (e.g., Chinese cabbage) , rapeseed, brassica, watermelon, melon, potato, tomato, tobacco, eggplant, pepper, cucumber, cotton, alfalfa, eggplant, grape.
  • the plant is a monocotyledon.
  • the monocotyledon is selected from the group consisting of rice, corn, wheat, barley, oat, sorghum, millet, grasses, Poaceae, Zizania, Avena, Coix, Hordeum, Oryza, Panicum (e.g., Panicum miliaceum) , Secale, Setaria (e.g., Setaria italica) , Sorghum, Triticum, Zea, Cymbopogon, Saccharum (e.g., Saccharum officinarum) , Phyllostachys, Dendrocalamus, Bambusa, Yushania.
  • the animal is selected from the group consisting of pig, ox, sheep, goat, mouse, rat, alpaca, monkey, rabbit, chicken, duck, goose, fish (e.g., zebra fish) .
  • the disclosure provides a cell modified by the method of the disclosure.
  • the method of the disclosure can be used to introduce the system of the disclosure into a cell and cause the cell to alter the production of one or more cellular produces, such as antibody, starch, ethanol, or any other desired products.
  • cellular produces such as antibody, starch, ethanol, or any other desired products.
  • Such cells and progenies thereof are within the scope of the disclosure.
  • the disclosure provides a cell comprising the system of the disclosure.
  • the cell is a eukaryotic cell.
  • the cell is a human cell.
  • the disclosure provides a cell modified by the system of the disclosure or the method of the disclosure.
  • the cell is a eukaryotic cell.
  • the cell is a human cell.
  • the cell is modified in vitro, in vivo, or ex vivo.
  • the cell is a stem cell. In some embodiments, the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell (e.g., an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., mouse or rat) cell, a human cell, a plant cell, or a yeast cell) or a prokaryotic cell (e.g., a bacteria cell) .
  • a eukaryotic cell e.g., an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., mouse or rat) cell, a human cell, a plant cell, or a yeast cell
  • a prokaryotic cell e.g., a bacteria cell
  • the cell is from a plant or an animal.
  • the plant is a dicotyledon.
  • the dicotyledon is selected from the group consisting of soybean, cabbage (e.g., Chinese cabbage) , rapeseed, brassica, watermelon, melon, potato, tomato, tobacco, eggplant, pepper, cucumber, cotton, alfalfa, eggplant, grape.
  • the plant is a monocotyledon.
  • the monocotyledon is selected from the group consisting of rice, corn, wheat, barley, oat, sorghum, millet, grasses, Poaceae, Zizania, Avena, Coix, Hordeum, Oryza, Panicum (e.g., Panicum miliaceum) , Secale, Setaria (e.g., Setaria italica) , Sorghum, Triticum, Zea, Cymbopogon, Saccharum (e.g., Saccharum officinarum) , Phyllostachys, Dendrocalamus, Bambusa, Yushania.
  • the animal is selected from the group consisting of pig, ox, sheep, goat, mouse, rat, alpaca, monkey, rabbit, chicken, duck, goose, fish (e.g., zebra fish) .
  • the cell is a eukaryotic cell, such as a mammalian cell, including a human cell (aprimary human cell or an established human cell line) .
  • the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey) , a cow /bull /cattle, sheep, goat, pig, horse, dog, cat, rodent (such as rabbit, mouse, rat, hamster, etc. ) .
  • the cell is from fish (such as salmon) , bird (such as poultry bird, including chick, duck, goose) , reptile, shellfish (e.g., oyster, claim, lobster, shrimp) , insect, worm, yeast, etc.
  • the cell is from a plant, such as monocot or dicot.
  • the plant is a food crop such as barley, cassava, cotton, groundnuts or peanuts, maize, millet, oil palm fruit, potatoes, pulses, rapeseed or canola, rice, rye, sorghum, soybeans, sugar cane, sugar beets, sunflower, and wheat.
  • the plant is a cereal (barley, maize, millet, rice, rye, sorghum, and wheat) .
  • the plant is a tuber (cassava and potatoes) .
  • the plant is a sugar crop (sugar beets and sugar cane) .
  • the plant is an oil-bearing crop (soybeans, groundnuts or peanuts, rapeseed or canola, sunflower, and oil palm fruit) .
  • the plant is a fiber crop (cotton) .
  • the plant is a tree (such as a peach or a nectarine tree, an apple or pear tree, a nut tree such as almond or walnut or pistachio tree, or a citrus tree, e.g., orange, grapefruit or lemon tree) , a grass, a vegetable, a fruit, or an algae.
  • a tree such as a peach or a nectarine tree, an apple or pear tree, a nut tree such as almond or walnut or pistachio tree, or a citrus tree, e.g., orange, grapefruit or lemon tree
  • the plant is a nightshade plant; a plant of the genus Brassica; a plant of the genus Lactuca; a plant of the genus Spinacia; a plant of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising (1) the system of the disclosure, the vector of the disclosure, the rAAV particle of the disclosure, the ribonucleoprotein of the disclosure, the lipid nanoparticle of the disclosure, or the cell of the disclosure; and (2) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises the rAAV particle in a concentration selected from the group consisting of about 1 ⁇ 10 10 vg/mL, 2 ⁇ 10 10 vg/mL, 3 ⁇ 10 10 vg/mL, 4 ⁇ 10 10 vg/mL, 5 ⁇ 10 10 vg/mL, 6 ⁇ 10 10 vg/mL, 7 ⁇ 10 10 vg/mL, 8 ⁇ 10 10 vg/mL, 9 ⁇ 10 10 vg/mL, 1 ⁇ 10 11 vg/mL, 2 ⁇ 10 11 vg/mL, 3 ⁇ 10 11 vg/mL, 4 ⁇ 10 11 vg/mL, 5 ⁇ 10 11 vg/mL, 6 ⁇ 10 11 vg/mL, 7 ⁇ 10 11 vg/mL, 8 ⁇ 10 11 vg/mL, 9 ⁇ 10 11 vg/mL, 1 ⁇ 10 12 vg/mL, 2 ⁇ 10 12 vg/mL, 3 ⁇ 10 12 vg/
  • the pharmaceutical composition is an injection.
  • the volume of the injection is selected from the group consisting of about 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, 350 microliters, 400 microliters, 450 microliters, 500 microliters, 550 microliters, 600 microliters, 650 microliters, 700 microliters, 750 microliters, 800 microliters, 850 microliters, 900 microliters, 950 microliters, 1000 microliters, and a volume of a numerical range between any of two preceding values, e.g., in a concentration of from about 10 microliters to about 750 microliters.
  • the disclosure provides a method for diagnosing, preventing, or treating a disease in a subject in need thereof, comprising administering to the subject the system of the disclosure, the vector of the disclosure, the rAAV particle of the disclosure, the ribonucleoprotein of the disclosure, the lipid nanoparticle of the disclosure, the cell of the disclosure, or the pharmaceutical composition of the disclosure, wherein the disease is associated with a target DNA, wherein the guide sequence is capable of hybridizing to a target sequence of the target DNA, wherein the target DNA is modified by the complex, and wherein the modification of the target DNA diagnose, prevents, or treats the disease.
  • the disease is selected from the group consisting of Angelman syndrome (AS) , Alzheimer's disease (AD) , transthyretin amyloidosis (ATTR) , transthyretin amyloid cardiomyopathy (ATTR-CM) , cystic fibrosis (CF) , hereditary angioedema, diabetes, progressive pseudohypertrophic muscular dystrophy, Duchenne muscular dystrophy (DMD) , Becker muscular dystrophy (BMD) , spinal muscular atrophy (SMA) , alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington’s disease (HTT) , fragile X syndrome, Friedreich ataxia, amyotrophic lateral sclerosis (ALS) , frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, Leber congenital amaurosis (LCA) , sickle cell disease, thalassemia (e.g., ⁇ -thalassemia)
  • the target DNA encodes a mRNA, a tRNA, a ribosomal RNA (rRNA) , a microRNA (miRNA) , a non-coding RNA, a long non-coding (lnc) RNA, a nuclear RNA, an interfering RNA (iRNA) , a small interfering RNA (siRNA) , a ribozyme, a riboswitch, a satellite RNA, a microswitch, a microzyme, or a viral RNA.
  • iRNA interfering RNA
  • siRNA small interfering RNA
  • the target DNA is a eukaryotic DNA.
  • the eukaryotic DNA is a mammal DNA, such as a non-human mammalian DNA, a non-human primate DNA, a human DNA, a plant DNA, an insect DNA, a bird DNA, a reptile DNA, a rodent (e.g., mouse, rat) DNA, a fish DNA, a nematode DNA, or a yeast DNA.
  • a mammal DNA such as a non-human mammalian DNA, a non-human primate DNA, a human DNA, a plant DNA, an insect DNA, a bird DNA, a reptile DNA, a rodent (e.g., mouse, rat) DNA, a fish DNA, a nematode DNA, or a yeast DNA.
  • the target DNA is in a eukaryotic cell, for example, a human cell, a non-human primate cell, or a mouse cell.
  • the administrating comprises local administration or systemic administration.
  • the administrating comprises intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotaxic administration.
  • the administration is injection or infusion.
  • the subject is a human, a non-human primate, or a mouse.
  • the level of the transcript (e.g., mRNA) of the target DNA is decreased in the subject by at least about 10%, about 15%, about 20%, about 25%, about 5%, 10%, 15%, 20%, 25%, 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of the transcript (e.g., mRNA) of the target DNA in the subject prior to the administration.
  • the level of the transcript (e.g., mRNA) of the target DNA is increased in the subject by at least about 10%, about 15%, about 20%, about 25%, about 5%, 10%, 15%, 20%, 25%, 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of the transcript (e.g., mRNA) of the target DNA in the subject prior to the administration.
  • the level of the expression product (e.g., protein) of the target DNA is decreased in the subject by at least about 10%, about 15%, about 20%, about 25%, about 5%, 10%, 15%, 20%, 25%, 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of the expression product (e.g., protein) of the target DNA in the subject prior to the administration.
  • the level of the expression product (e.g., protein) of the target DNA is increased in the subject by at least about 10%, about 15%, about 20%, about 25%, about 5%, 10%, 15%, 20%, 25%, 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of the expression product (e.g., protein) of the target DNA in the subject prior to the administration.
  • the expression product is a functional mutant of the expression product of the target DNA.
  • the median survival of the subject suffering from the disease but receiving the administration is 5 days, 10 days, 20 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 year, 2 years, 2.5 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more longer than that of a subject or a population of subjects suffering from the disease and not receiving the administration.
  • the therapeutically effective dose may be either via a single dose, or multiple doses.
  • the actual dose may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.
  • the therapeutically effective dose of the rAAV particle may be about 1.0E+8, 2.0E+8, 3.0E+8, 4.0E+8, 6.0E+8, 8.0E+8, 1.0E+9, 2.0E+9, 3.0E+9, 4.0E+9, 6.0E+9, 8.0E+9, 1.0E+10, 2.0E+10, 3.0E+10, 4.0E+10, 6.0E+10, 8.0E+10, 1.0E+11, 2.0E+11, 3.0E+11, 4.0E+11, 6.0E+11, 8.0E+11, 1.0E+12, 2.0E+12, 3.0E+12, 4.0E+12, 6.0E+12, 8.0E+12, 1.0E+13, 2.0E+13, 3.0E+13, 4.0E+13, 6.0E+13, 8.0E+13, 1.0E+14, 2.0E+14, 3.0E+14, 4.0E+14, 6.0E+14, 8.0E+14, 1.0E+15, 2.0E+15, 2.0
  • the disclosure provides a method of detecting a target DNA, comprising contacting the target DNA with the system of the disclosure.
  • the target DNA is modified by the system, and wherein the modification detects the target DNA.
  • the modification generates a detectable signal, e.g., a fluorescent signal.
  • the disclosure provides a kit comprising the IscB polypeptide of the disclosure, the system of the disclosure, the polynucleotide of the disclosure, the vector of the disclosure, the RNP of the disclosure, the LNP of the disclosure, the delivery system of the disclosure, the cell of the disclosure, or the pharmaceutical composition of the disclosure, or any one, two, or all components of the same.
  • the kit further comprises an instruction to use the component (s) contained therein, and/or instructions for combining with additional component (s) that may be available or necessary elsewhere.
  • the kit further comprises one or more buffers that may be used to dissolve any of the component (s) contained therein, and/or to provide suitable reaction conditions for one or more of the component (s) .
  • buffers may include one or more of PBS, HEPES, Tris, MOPS, Na 2 CO 3 , NaHCO 3 , NaB, or combinations thereof.
  • the reaction condition includes a proper pH, such as a basic pH. In some embodiments, the pH is between 7-10.
  • any one or more of the kit components may be stored in a suitable container or at a suitable temperature, e.g., 4 degree Celsius.
  • the disclosure provides an IscB nickase comprising an amino acid substitution of:
  • x is any amino acid residue
  • the IscB nickase substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially retains ssDNA cleavage activity (e.g., has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more ssDNA cleavage activity of the corresponding wild-type IscB) on the target strand of the target dsDNA.
  • dsDNA cleavage activity e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%,
  • the disclosure provides an IscB nickase comprising an amino acid substitution of:
  • x is any amino acid residue
  • the IscB nickase substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially retains ssDNA cleavage activity (e.g., has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more ssDNA cleavage activity of the corresponding wild-type IscB) on the non-target strand of the target dsDNA.
  • dsDNA cleavage activity e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%,
  • the disclosure provides an IscB nickase of an IscB, comprising a deletion of one or more (or all) amino acid residues of the HNH domain of the IscB, wherein the IscB nickase substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially retains ssDNA cleavage activity (e.g., has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more ssDNA cleavage activity of the corresponding wild-type IscB) on the non-target
  • the disclosure provides an endonuclease-deficient IscB (endonuclease-deficient IscB or dIscB) comprising:
  • x is any amino acid residue
  • the endonuclease-deficient IscB substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially lacks ssDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less ssDNA cleavage activity of the corresponding wild-type IscB) on either the non-target strand or target strand of the target dsDNA.
  • dsDNA cleavage activity e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%,
  • the substitution is a substitution with a non-polar amino acid residue, a polar amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue.
  • the non-polar amino acid residue is selected from the group consisting of Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ; optionally Alanine (Ala/A) .
  • the polar amino acid residue is selected from the group consisting of Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) .
  • the positively charged amino acid residue is selected from the group consisting of Lysine (Lys/K) , Arginine (Arg/R) , and Histidine (His/H) ; optionally Arginine (Arg/R) .
  • the negatively charged amino acid residue is selected from the group consisting of Aspartic Acid (Asp/D) and Glutamic Acid (Glue/E) ) .
  • the D of motif DxG in RuvC I domain is at a position corresponding to D61 of SEQ ID NO: 24.
  • the G of motif DxG in RuvC I domain is at a position corresponding to G63 of SEQ ID NO: 24.
  • the E of motif ExxxF in RuvC II domain is at a position corresponding to E193 of SEQ ID NO: 24.
  • the F of motif ExxxF in RuvC II domain is at a position corresponding to F197 of SEQ ID NO: 24.
  • the H of motif HxxDA in RuvC III domain is at a position corresponding to H340 of SEQ ID NO: 24.
  • the D of motif HxxDA in RuvC III domain is at a position corresponding to D343 of SEQ ID NO: 24.
  • the A of motif HxxDA in RuvC III domain is at a position corresponding to A344 of SEQ ID NO: 24.
  • the H 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in HNH domain is at a position corresponding to H248 of SEQ ID NO: 24.
  • the H 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 ⁇ in HNH domain is at a position corresponding to H271 of SEQ ID NO: 24.
  • the H 3 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 ⁇ in HNH domain is at a position corresponding to H275 of SEQ ID NO: 24.
  • the N 2 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ N 2 xxK 1 ⁇ in HNH domain is at a position corresponding to N236 of SEQ ID NO: 47.
  • the K 1 of motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ N 2 xxK 1 ⁇ in HNH domain is at a position corresponding to K239 of SEQ ID NO: 47.
  • the IscB nickase or endonuclease-deficient IscB has a sequence identity of at least about 50% (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 21-86.
  • the IscB nickase is not AwaIscB-E157A.
  • the IscB nickase is not AwaIscB-H212A.
  • the endonuclease-deficient IscB is not AwaIscB-E157A, H212A.
  • the disclosure provides a fusion protein or conjugate comprising the IscB nickase or endonuclease-deficient IscB of the disclosure and a functional domain.
  • the functional domain is directly or indirectly, covalently or non-covalently linked (e.g., fused) to IscB nickase or endonuclease-deficient IscB or adapted to link thereto after delivery.
  • the functional domain is directly or indirectly, covalently or non-covalently linked (e.g., fused) to IscB nickase or endonuclease-deficient IscB or adapted to link thereto after delivery.
  • the functional domain is directly or indirectly, covalently or non-covalently linked (e.g., fused) N-terminally, C-terminally, or internally with respect to IscB nickase or endonuclease-deficient IscB.
  • the functional domain is selected from the group consisting of an exonuclease, a reverse transcriptase (such as, Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) ) , a nuclear localization signal (NLS) , a nuclear export signal (NES) , a deaminase or a catalytic domain thereof, an uracil glycosylase inhibitor (UGI) , an uracil glycosylase (UNG) , a methylpurine glycosylase (MPG) , a methylase or a catalytic domain thereof, a demethylase or a catalytic domain thereof, an transcription activating domain (e.g., VP64 or VPR) , an transcription inhibiting domain (e.g., KRAB moiety or SID moiety) , a histone residue modification domain, a nuclease catalytic domain (e.g., FokI)
  • the NLS comprises or is SV40 NLS (such as SEQ ID NO: 13) or NP NLS (such as SEQ ID NO: 14) ;
  • the functional domain is a deaminase or catalytic domain thereof
  • the deaminase or catalytic domain thereof is selected from a group consisting of an adenosine deaminase (e.g., TadA, such as, TadA8e, TadA8.17, TadA8.20, TadA9) and a catalytic domain thereof, a cytidine deaminase (e.g., APOBEC, such as, APOBEC3, for example, APOBEC3A, APOBEC3B, APOBEC3C; DddA) and a catalytic domain thereof, a bifunctional adenosine &cytidine deaminase and a catalytic domain thereof, and a functional fragment thereof, and a variant thereof, and any combination thereof.
  • an adenosine deaminase e.g., TadA, such as, TadA8e, TadA8.17, TadA8.20, TadA9
  • the functional domain is an uracil glycosylase inhibitor (UGI) .
  • UMI uracil glycosylase inhibitor
  • the functional domain is an uracil glycosylase (UNG) .
  • UNG uracil glycosylase
  • the functional domain is a methylpurine glycosylase (MPG) .
  • MPG methylpurine glycosylase
  • the functional domain is a methylase or a catalytic domain thereof.
  • the functional domain is a transcription activating domain.
  • the functional domain is an exonuclease, such as, T5 exonuclease (T5E) .
  • T5E T5 exonuclease
  • the exonuclease is N-terminally or C-terminally fused to the IscB nickase or endonuclease-deficient IscB.
  • the exonuclease is C-terminally fused to the IscB nickase or endonuclease-deficient IscB.
  • an IscB system comprising:
  • IscB nickase or endonuclease-deficient IscB of the disclosure or the fusion protein or conjugate of the disclosure and
  • the scaffold sequence has a sequence identity of at least about 50% (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%) to the nucleotide sequence of any one of SEQ ID NOs: 87-152.
  • the scaffold sequence has substantially the same secondary structure of any one of SEQ ID NOs: 87-152.
  • the secondary structure of the scaffold sequence can be precited and depicted by any known method in the art.
  • an online tool http: //rna. tbi. univie. ac. at/cgi-bin/RNAWebSuite/RNAfold. cgi)
  • RNAfold http: //rna. tbi. univie. ac. at/cgi-bin/RNAWebSuite/RNAfold. cgi
  • the option “minimum free energy (MFE) and partition function” is selected for “Fold algorithms and basic options” . The other parameters are default. After the results for minimum free energy prediction are outputted, “MFE plain structure drawing” is selected for image description and comparing.
  • substantially the same secondary structure means that the two secondary structures have the same number of stem-loops.
  • the disclosure provides a method of modifying a target dsDNA comprising contacting the target dsDNA with the IscB system of the disclosure.
  • the disclosure provides a method of identifying IscB nickase comprising:
  • identifying the IscB polypeptide substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially retains ssDNA cleavage activity (e.g., has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more ssDNA cleavage activity of the corresponding wild-type IscB) on the target strand of the target dsDNA.
  • dsDNA cleavage activity e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%,
  • the disclosure provides a method of identifying IscB nickase comprising:
  • identifying the IscB polypeptide substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially retains ssDNA cleavage activity (e.g., has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more ssDNA cleavage activity of the corresponding wild-type IscB) on the non-target strand of the target dsDNA.
  • dsDNA cleavage activity e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%,
  • the disclosure provides a method of identifying dead IscB comprising:
  • identifying the IscB polypeptide substantially lacks dsDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on a target dsDNA and substantially lacks ssDNA cleavage activity (e.g., has at most about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less dsDNA cleavage activity of the corresponding wild-type IscB) on either the target strand or the non-target strand of the target dsDNA.
  • Example 1 Design of IscB substantially lacking endonuclease activity and evaluation of endonuclease and nickase activities thereof.
  • This Example demonstrates the design of IscB substantially lacking endonuclease activity and evaluation of endonuclease and nickase activities thereof.
  • the motif was composed of conserved amino acid residue H 1 (where “1” means that it is the first H in the motif) and conserved amino acid residue N 1 (where “1” means that it is the first N in the motif) and 12 to 17 undefined amino acid residues x between H 1 and N 1 (shown as “ ⁇ 12-17x ⁇ ” ) , followed by 8 undefined amino acid residues x (shown as “ ⁇ 8x ⁇ ” ) and further by one of three different conditions:
  • conserved amino acid residue it means that the amino acid residue is constant (not changed) as indicated across all the analyzed IscB.
  • conserve motif it means that the motif has a constant (not changed) pattern as indicated across all the analyzed IscB. See “CLUSTAL multiple sequence alignment by Kalign (3.3.1) ” in the “EXAMPLARY SEQUENCES” section of the disclosure.
  • HISCB016 (SEQ ID NO: 29) as an example, it contains motif P in RuvC I domain, motif INK in RuvC II domain, and motif HI in RuvC III domain, and motif VIPKHEHGSDTIA IVGLCSGC DLV in HNH domain (the conserved amino acids are double-underlined) .
  • HISCB016 SEQ ID NO: 29
  • HISCB017 SEQ ID NO: 30
  • OgeuIscB SEQ ID NO: 89
  • enOgeuIscB SEQ ID NO: 158
  • IscB polypeptides for development of engineered IscB polypeptides (mutants) substantially lacking endonuclease activity.
  • FIG. 1 a dual plasmid GFxxFP reporter system was established (FIG. 1) , containing an expression plasmid, and either a reporter plasmid for endonuclease activity evaluation or a reporter plasmid for nickase activity evaluation.
  • the expression plasmid (the upper construct in FIG. 1) comprised, from 5’ to 3’, (1) a polynucleotide encoding an IscB polypeptide flanked by N-terminal SV40 NLS (SEQ ID NO: 21) and C-terminal NP NLS (SEQ ID NO: 22) operably linked to CAG promoter (SEQ ID NO: 18) followed by bGH polyA signal (SEQ ID NO: 20) ; (2) a polynucleotide encoding a guide nucleic acid operably linked to U6 promoter (SEQ ID NO: 19) ; and (3) a polynucleotide encoding mCherry (SEQ ID NO: 16) operably linked to CMV promoter (SEQ ID NO: 17) followed by bGH polyA signal (SEQ ID NO: 20) . Red fluorescent signals generated by the expression of the mCherry indicated successful transfection and expression of the expression plasmid in host cells.
  • the two reporter plasmids comprised a polynucleotide encoding BFP-T2A-GFxxFP expression cassette with a deactivated EGFP coding sequence (GFxxFP coding sequence) operably linked to CMV promoter (SEQ ID NO: 17) followed by bGH polyA signal (SEQ ID NO: 20) .
  • Blue fluorescent signals generated by the expression of the BFP indicated successful transfection and expression of the reporter plasmid in host cells.
  • the GFxxFP coding sequence (SEQ ID NO: 1) harbored an insertion sequence (SEQ ID NO: 2) containing protospacer sequence (SEQ ID NO: 6) containing premature stop codon , immediately5’ to a target adjacent motif (TAM) , on the 5’-3’ strand, which premature stop codon prevents translation of the GFxxFP coding sequence.
  • the TAM can be changed for any TAM needed for the evaluation of endonuclease activity of any selected IscB polypeptide.
  • the TAM is 5’- -3’ (SEQ ID NO: 7)
  • the TAM is replaced with 5’- -3’ (SEQ ID NO: 8)
  • the insertion sequence SEQ ID NO: 2 was replaced with insertion sequence (SEQ ID NO: 25) containing the protospacer sequence (SEQ ID NO: 6)
  • the TAM is 5’- -3’ (SEQ ID NO: 7) . See the middle construct in FIG. 1.
  • the GFxxFP coding sequence (SEQ ID NO: 3) harbored an insertion sequence (SEQ ID NO: 4) containing (1) the complement (SEQ ID NO: 9) of protospacer sequence containing premature stop codon , immediately 5’ to a TAM, on the 3’-5’ strand and (2) protospacer sequence (SEQ ID NO: 6) containing premature stop codon , immediately 5’ to a TAM, on the 5’-3’ strand, which premature stop codon prevents translation of the GFxxFP coding sequence.
  • the GFxxFP coding sequence contained a 12 nt linker (SEQ ID NO: 168) between the complement (SEQ ID NO: 9) of protospacer sequence and the protospacer sequence (SEQ ID NO: 6) .
  • the TAM can be changed for any TAM needed for the evaluation of nickase activity of any selected IscB polypeptide.
  • the TAM on the 5’-3’ strand is 5’- -3’ (SEQ ID NO: 7)
  • the TAM on the 5’-3’ strand is replaced with 5’- -3’ (SEQ ID NO: 8) .
  • the TAM on the 3’-5’ strand is 3’ -5’ (SEQ ID NO: 10)
  • the TAM on the 3’-5’ strand is replaced with 3’- -5’ (SEQ ID NO: 11) .
  • the insertion sequence (SEQ ID NO: 4) was replaced with insertion sequence (SEQ ID NO: 12) containing the complement (SEQ ID NO: 9) of protospacer sequence and the protospacer sequence (SEQ ID NO: 6) , the TAM on the 5’ -3’ strand is 5’- -3’ (SEQ ID NO: 14) , and the TAM on the 3’-5’ strand is 3’- -5’ (SEQ ID NO: 15) . See the lower construct in FIG. 1.
  • the guide nucleic acid was designed to target the insertion sequence of the GFxxFP coding sequence so as to trigger double-strand cleavage or single-strand cleavage (nick) at the insertion sequence comprising the protospacer sequence and/or the complement of the protospacer sequence.
  • the guide nucleic acid was an RNA (termed as omega RNA ( ⁇ RNA) or guide RNA (gRNA) as used herein) composed of a guide sequence immediately 5’ to a scaffold sequence (or termed as ⁇ RNA scaffold sequence) .
  • ⁇ RNA omega RNA
  • gRNA guide RNA
  • the guide sequence is GFxxFP-targeting guide sequence (SEQ ID NO: 23) capable of targeting the insertion sequence.
  • the guide sequence is nontargeting guide sequence (SEQ ID NO: 24) incapable of targeting the insertion sequence.
  • the scaffold sequence is HISCB016 scaffold sequence (SEQ ID NO: 95) for HISCB016 or mutants thereof, HISCB017 scaffold sequence (SEQ ID NO: 96) for HISCB017 or mutants thereof, OgeuIscB scaffold sequence (SEQ ID NO: 155) for OgeuIscB or mutants thereof, or enOgeuIscB scaffold sequence (SEQ ID NO: 159) for enOgeuIscB or mutants thereof.
  • the GFxxFP-targeting ⁇ RNA was SEQ ID NO: 160 (where “t” denotes “u” for RNA sequences) for HISCB016 or mutants thereof, SEQ ID NO: 161 (where “t” denotes “u” for RNA sequences) for HISCB017 or mutants thereof, SEQ ID NO: 162 (where “t” denotes “u” for RNA sequences) for OgeuIscB or mutants thereof, and SEQ ID NO: 163 (where “t” denotes “u” for RNA sequences) for enOgeuIscB or mutants thereof.
  • the reporter plasmid for endonuclease activity evaluation (FIG. 1, the middle construct)
  • the subsequent DNA repairing trigged by the DSB would restore and activate the deactivated EGFP coding sequence to express EGFP with green fluorescence emission indicative of endonuclease activity functioning in a guide sequence-specific (on-target) manner.
  • the reporter plasmid for nickase activity evaluation (FIG. 1, the lower construct)
  • the two SSB are similar to a DSB and may also trigger subsequent DNA repairing, thereby restoring and activating the deactivated EGFP coding sequence to express EGFP with green fluorescence emission indicative of nickase activity functioning in a guide sequence-specific (on-target) manner.
  • the tested wild-type IscB polypeptides (HISCB016, HISCB017, and OgeuIscB) , while showing double-strand cleavage demonstrating that they are endonucleases, may also show single-strand cleavage (since certainly the endonuclease activity can generate SSB) in the nickase evaluation reporter system, which fact however does not make them nickase.
  • HEK293T cells were cultured in 24-well tissue culture plates according to standard methods for 12 hours, before the reporter and expression plasmids were co-transfected into the cells using standard polyethyleneimine (PEI) transfection. The transfected cells were then cultured at 37°C under 5%CO 2 for 48 hours. Then the cultured cells were analyzed by flow cytometry for BFP, EGFP, and mCherry fluorescent signals.
  • PEI polyethyleneimine
  • the endonuclease and nickase activities were calculated as the percentage of EGFP positive cells ( “EGFP + ” ) in BFP &mCherry dual-positive cells ( “mCherry + BFP + ” ) .
  • H245A and H245R mutations which locate in the HNH domain of HISCB016 but not locate in the defined motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain, still showed endonuclease activity (FIG. 2) , suggesting that it might be the mutagenesis within the motif that matters, not just mutagenesis within the domain.
  • a dead HISCB016 mutant ( “DEAD” ; dead HISCB016 or dHISCB016) (e.g., HISCB016-E193A+H248A) was generated, lacking both endonuclease activity and nickase activity.
  • H241A and H241R mutations which locate in the HNH domain of HISCB017 but not locate in the defined motif H 1 ⁇ 12-17x ⁇ N 1 ⁇ 8x ⁇ ⁇ H 2 xxxH 3 /H 2 xxxxH 3 /N 2 xxK 1 ⁇ in the HNH domain of HISCB017, still showed endonuclease activity (FIG. 4) , suggesting that it might be the mutagenesis within the motif that matters, not just mutagenesis within the domain.
  • a dead HISCB017 ( “DEAD” ; dead HISCB017 or dHISCB017) (e.g., HISCB017-E190A+H244A) was generated, lacking both endonuclease activity and nickase activity.
  • This Example demonstrates additional designs of IscB substantially lacking endonuclease activity and evaluation of endonuclease and nickase activities thereof.
  • amino acids are classified into four types of:
  • non-polar amino acids including Glycine (Gly/G) , Alanine (Ala/A) , Valine (Val/V) , Cysteine (Cys/C) , Proline (Pro/P) , Leucine (Leu/L) , Isoleucine (Ile/I) , Methionine (Met/M) , Tryptophan (Trp/W) , and Phenylalanine (Phe/F) ;
  • polar amino acids including Serine (Ser/S) , Threonine (Thr/T) , Tyrosine (Tyr/Y) , Asparagine (Asn/N) , and Glutamine (Gln/Q) ; and
  • Lysine Lys/K
  • Arginine Arg/R
  • Histidine Histidine
  • a substitution of an amino acid with another amino acid of the same type is termed as a conservative substitution
  • a substitution of an amino acid with another amino acid of a different type is termed as a non-conservative substitution.
  • a representative amino acid was selected from each of the four types for substitution at the indicated position. Specifically, Alanine (A) was selected to represent non-polar amino acids for substitution except for position A344 of HISCB016, position A337 of HISCB017, and position A344 of OgeuIscB where Glycine (Gly/G) was selected instead; Glutamic Acid (Glu/E) was selected to represent negatively charged amino acids except for position E193 of HISCB016, position E190 of HISCB017, and position E193 of OgeuIscB where Aspartic Acid (Asp/D) was selected instead; Glutamine (Gln/Q) was selected to represent polar amino acids; and Arginine (Arg/R) was selected to represent positively charged amino acids.
  • Alanine (A) was selected to represent non-polar amino acids for substitution except for position A344 of HISCB016, position A337 of HISCB017, and position A344 of OgeuIsc
  • This Example demonstrates endonuclease activity of additional IscB polypeptides in Table 7 in eukaryotic cells.

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Abstract

L'invention concerne des polypeptides IscB destinés à diverses applications, par exemple, le clivage à double brin, l'édition de base, l'édition primaire.
PCT/CN2023/125069 2022-10-17 2023-10-17 Polypeptides iscb et leurs utilisations Ceased WO2024083135A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022087494A1 (fr) * 2020-10-23 2022-04-28 The Broad Institute, Inc. Nucléases iscb reprogrammables et leurs utilisations
WO2022159892A1 (fr) * 2021-01-25 2022-07-28 The Broad Institute, Inc. Polypeptides tnpb reprogrammables et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022087494A1 (fr) * 2020-10-23 2022-04-28 The Broad Institute, Inc. Nucléases iscb reprogrammables et leurs utilisations
WO2022159892A1 (fr) * 2021-01-25 2022-07-28 The Broad Institute, Inc. Polypeptides tnpb reprogrammables et leur utilisation

Non-Patent Citations (4)

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Title
DATABASE Protein 18 February 2015 (2015-02-18), ANONYMOUS: "Sequence 3046 from patent US 8569030", XP093160448, retrieved from NCBI Database accession no. AJO39039.1 *
DATABASE Protein 21 April 2021 (2021-04-21), ANONYMOUS: "MAG: HNH endonuclease [Lachnospiraceae bacterium]", XP093160446, retrieved from NCBI Database accession no. MBQ7557675.1 *
DATABASE UniProtKB/TrEMBL 13 February 2019 (2019-02-13), ANONYMOUS: "Full=Uncharacterized protein {ECO:0000313|EMBL:BBH25940.1}", XP093160449, retrieved from UniProt Database accession no. A0A3G9J454 *
GABRIEL SCHULER: "Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 376, no. 6600, 24 June 2022 (2022-06-24), US , pages 1476 - 1481, XP093160450, ISSN: 0036-8075, DOI: 10.1126/science.abq7220 *

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