S. pyogenes CAS9 mutant genes and polypeptides encoded by same

A CRISPR/Cas endonuclease system comprising:

• mutant Cas9 protein comprising 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:
• F682A, and K684A;
• or
• (b) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO:
• 5 selected from two of following positions:
• N690A, F682A, L683A, K684A and D686A;
• or
• (c) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO:
• 5 comprising any one of following positions:
• N690A, F682A, L683A, K684A and D686A in combination with any one of the following positions:
• R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925;
• and
• a gRNA,
• wherein the resultant CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity for at least one target site relative to a wild-type CRISPR/Cas endonuclease system having a WT-Cas9 protein of SEQ ID NO:5.

An isolated nucleic acid encoding an isolated mutant Cas9 protein comprising 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:
• F682A, and K684A;
• or
• (b) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO:
• 5 selected from two of following positions:
• N690, F682, L683, K684 and D686;
• or
• (c) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO:
• 5 comprising any one of following positions:
• N690, F682, L683, K684 and D686 in combination with any one of the following positions:
• R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925,
• wherein the isolated mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the resultant CRISPR/Cas endonuclease system displays a lower ratio of reduced off-target editing activity to on-target editing activity for at least one target site relative to the corresponding ratio of reduced off-site editing activity to on-target editing activity of a wild-type CRISPR/Cas endonuclease system having a WT-Cas9 protein of SEQ ID NO:5.