CPC C12N 15/11 (2013.01) [C12N 9/22 (2013.01); C12N 15/902 (2013.01); C12N 15/113 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] | 7 Claims |
1. A nucleic acid vector encoding an isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays a lower ratio of off-target editing activity to on-target editing activity for at least one target relative to the corresponding ratio of off-target editing activity to on-target editing activity of a wild-type CRISPR/Cas endonuclease system having the wild-type Cas9 protein of SEQ ID NO: 5,
wherein the isolated mutant Cas9 protein comprises a substitution mutation selected from the group consisting of
(a) a single substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO: 5 selected from the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, S793, N803, N854, S872 and R925; or
(b) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO: 5 selected from two of following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925.
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