S. pyogenes CAS9 mutant genes and polypeptides encoded by same
This invention pertains to isolated mutant Cas9 nucleic acids and proteins for use in CRISPR/Cas endonuclease systems and their methods of use. In particular, the invention pertains to 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 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.
1 . An isolated ribonucleoprotein complex, comprising:
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: 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 complex,
wherein the isolated ribonucleoprotein complex is active as a CRISPR/Cas endonuclease system, 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.
2 . The isolated ribonucleoprotein complex of claim 1 , wherein the gRNA comprises a crRNA and a tracrRNA in stoichiometric (1:1) ratio.
3 . The isolated ribonucleoprotein complex of claim 1 , wherein
the gRNA comprises an isolated crRNA: tracrRNA complex.
4 . The isolated ribonucleoprotein complex of claim 1 , wherein the gRNA comprises a sgRNA.
5 . The isolated ribonucleoprotein complex of claim 1 , wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NO: 143, and SEQ ID NO: 145.
6 . 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.
7 . The CRISPR/Cas endonuclease system of claim 6 , wherein CRISPR/Cas endonuclease system is encoded by a DNA expression vector.
8 . The CRISPR/Cas endonuclease system of claim 6 , wherein DNA expression vector comprises a plasmid-borne vector.
9 . The CRISPR/Cas endonuclease system of claim 6 , wherein DNA expression vector is selected from a bacterial expression vector and a eukaryotic expression vector.
10 . The CRISPR/Cas endonuclease system of claim 6 , wherein the gRNA includes a crRNA and a tracrRNA in stoichiometric (1:1) ratio.
11 . The CRISPR/Cas endonuclease system of claim 10 , wherein the gRNA comprising a crRNA: tracrRNA complex.
12 . The CRISPR/Cas endonuclease system of claim 10 , wherein the gRNA comprises a sgRNA.
13 . The CRISPR/Cas endonuclease system of claim 10 , wherein the mutant Cas9 protein comprises a substitution mutation selected from the group consisting of SEQ ID NO: 143, and SEQ ID NO: 145.
14 . A method of performing gene editing having 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, comprising:
contacting a candidate editing target site locus with an active CRISPR/Cas endonuclease system having the mutant Cas9 protein of claim 1 , 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.
15 . The method of claim 14 , wherein the mutant Cas9 protein comprises a substitution mutation selected from the group consisting of SEQ ID NO: 141-168.
16 . 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.
17 . The isolated nucleic acid of claim 16 , wherein the encoded mutant Cas9 protein is selected from the group consisting of SEQ ID NO: 141-168.