Engineered cas12b effector proteins and methods of use thereof
AssigneesBeijing Institute for Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences
InventorsWei LI, Qi ZHOU, Yangcan CHEN
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Abstract
Provided are engineered Cas12b nucleases or derivatives thereof comprising one or more types of mutations with improved activity (e.g, gene editing activity) or abolished nuclease activity. Also provided are engineered Cas12b effector proteins, engineered gRNAs (e.g, sgRNAs or tracrRNAs), engineered CRISPR-Cas12b systems, and methods of use thereof.
Claims29
An engineered Cas12b nuclease, comprising one, two, or three types of mutations with respect to a reference Cas12b nuclease, wherein the mutations comprise:
- (1) substitution of one or more amino acid residues in the reference Cas12b nuclease that interact with a protospacer adjacent motif (PAM) with a positively charged amino acid residue;
- and/or
- (2) substitution of one or more amino acid residues in the reference Cas12b nuclease that are involved in opening DNA double strands with an amino acid residue having an aromatic ring;
- and/or
- (3) substitution of one or more amino acid residues in the RuvC domain of the reference Cas12b nuclease that interact with a single-stranded DNA substrate with a positively charged amino acid residue or a hydrophobic amino acid residue.
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4-5. (canceled)
7-12. (canceled)
14-18. (canceled)
20-27. (canceled)
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34-38. (canceled)
40-42. (canceled)
A single guide RNA (sgRNA) comprising the sequence of any one of SEQ ID NOs:
- 25-53.
45-55. (canceled)
58-67. (canceled)
(canceled)
Dependencies are inferred from the claim text.