Patent
This invention pertains to 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 reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system. The invention also includes isolated nucleic acids encoding mutant Cas9 proteins, ribonucleoprotein complexes and CRISPR/Cas endonuclease systems having mutant Cas9 proteins that display reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.
1. An isolated mutant Cas9 protein consisting of a substitution mutation selected from the group consisting of
(a) 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
(b) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO:5 consisting of 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 mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.
2. An isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39-67.
3. The isolated mutant Cas9 protein of claim 1 , wherein the isolated mutant Cas9 protein consists of a double substitution mutation selected from the group consisting of N690A, F682A, L683A, K684A and D686A.
4. An isolated ribonucleoprotein complex, comprising:
(a) the mutant Cas9 protein of claim 1 ; and
(b) 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 relative to a wild-type CRISPR/Cas endonuclease system.
5. The isolated ribonucleoprotein complex of claim 4 , wherein the gRNA comprises a crRNA and a tracrRNA in stoichiometric (1:1) ratio.
6. The isolated ribonucleoprotein complex of claim 4 , wherein
(a) the isolated ribonucleoprotein complex comprises an isolated chemically synthesized crRNA:tracrRNA complex directed against a specific editing target site for a given locus.
7. The isolated ribonucleoprotein complex of claim 4 , wherein the gRNA comprises a sgRNA.
8. An isolated ribonucleoprotein complex, comprising:
(a) a mutant Cas9 protein; and
(b) 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 relative to a wild-type CRISPR/Cas endonuclease system,
wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39-67.
9. A CRISPR/Cas endonuclease system comprising the mutant Cas9 protein of claim 1 and a gRNA, wherein the CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.
10. The CRISPR/Cas endonuclease system of claim 9 , wherein CRISPR/Cas endonuclease system is encoded by a DNA expression vector.
11. The CRISPR/Cas endonuclease system of claim 10 , wherein DNA expression vector comprises a plasmid-borne vector.
12. The CRISPR/Cas endonuclease system of claim 10 , wherein DNA expression vector is selected from a bacterial expression vector and a eukaryotic expression vector.
13. The CRISPR/Cas endonuclease system of claim 9 , wherein the gRNA includes a crRNA and a tracrRNA in stoichiometric (1:1) ratio.
14. The CRISPR/Cas endonuclease system of claim 9 , wherein the CRISPR/Cas endonuclease system comprises a chemically synthesized crRNA:tracrRNA complex directed against a specific editing target site for a given locus.
15. The CRISPR/Cas endonuclease system of claim 9 , wherein the gRNA comprises a sgRNA.
16. A CRISPR/Cas endonuclease system, comprising:
(a) a mutant Cas9 protein; and
(b) a gRNA,
wherein the CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system,
wherein the mutant Cas9 protein is a polypeptide selected from the group consisting of SEQ ID NOs: 39-67.
17. A method of performing gene editing having reduced off-target editing activity and/or increased on-target editing activity, 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 active CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.
18. A method of performing gene editing having reduced off-target editing activity and/or increased on-target editing activity, comprising:
contacting a candidate editing target site locus with an active CRISPR/Cas endonuclease system having a mutant Cas9 protein, wherein the active CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system,
wherein the mutant Cas9 protein is selected from the group consisting of SEQ ID NOs: 39-67.