Engineered Cas9 nucleases
The present disclosure relates to Cas9 nuclease variants and methods of producing and using such variants.
1. A polypeptide having nuclease activity, comprising an amino acid sequence having at least 90% identity to SEQ ID NO:13, wherein the polypeptide comprises an amino acid substitution at two or more of the following positions of SEQ ID NO:13: D23, T67, Y128, and D1251.
2. The polypeptide of claim 1 , comprising an amino acid sequence having at least 95% identity to SEQ ID NO:13.
3. The polypeptide of claim 1 , comprising an amino acid sequence having at least 99% identity to SEQ ID NO:13.
4. The polypeptide of claim 1 , wherein the polypeptide comprises an amino acid substitution at three or more of the following positions of SEQ ID NO:13: D23, T67, Y128, and D1251.
5. The polypeptide of claim 1 , wherein the polypeptide comprises an amino acid substitution at all four of the following positions of SEQ ID NO:13: D23, T67, Y128, and D1251.
6. The polypeptide of claim 1 , wherein the polypeptide comprises three or more of the following amino acid substitutions relative to SEQ ID NO:13: D23A, T67L, Y128V and D1251G.
7. The polypeptide of claim 1 , wherein the polypeptide comprises the four following amino acid substitutions relative to SEQ ID NO:13: D23A, T67L, Y128V and D1251G.
8. An isolated ribonucleoprotein (RNP) complex comprising the polypeptide of claim 1 and a guide nucleic acid.
9. A polypeptide having nuclease activity, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13, wherein the polypeptide comprises two or more of the following amino acid substitutions relative to SEQ ID NO:13: D23A, T67L, Y128V and D1251G.
10. The polypeptide of claim 9 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 13 with the four following amino acid substitutions: D23A, Y67L, Y128V and D1251G.
11. A nucleic acid encoding a polypeptide having nuclease activity and comprising an amino acid sequence having at least 90% identity to SEQ ID NO:13, wherein the polypeptide comprises two or more of the following amino acid substitutions relative to SEQ ID NO:13: D23A, T67L, Y128V and D1251G.
12. The nucleic acid of claim 11 , wherein the polypeptide comprises three or more of the following amino acid substitutions relative to SEQ ID NO:13: D23A, T67L, Y128V and D1251G.
13. The nucleic acid of claim 11 , wherein the polypeptide comprises the four following amino acid substitutions relative to SEQ ID NO:13: D23A, T67L, Y128V and D1251G.
14. A vector comprising the nucleic acid of claim 11 .
15. A genome editing system comprising:
(i) a polypeptide having nuclease activity and comprising an amino acid sequence having at least 90% identity to SEQ ID NO:13, wherein the polypeptide comprises an amino acid substitution at two or more of the following positions of SEQ ID NO:13: D23, T67, Y128, and D1251; and
(ii) a guide nucleic acid.
16. The genome editing system of claim 15 , wherein the guide nucleic acid is a guide RNA.
17. The genome editing system of claim 15 , wherein upon administration of the genome editing system to a target cell comprising a target sequence and an off-target sequence, a rate of off-target editing of the off-target sequence by the genome editing system is less than an observed rate of off-target editing of the off-target sequence upon administration of a control genome editing system to the target cell, wherein the control genome editing system comprises:
a polypeptide having nuclease activity and comprising SEQ ID NO:13, and
the guide nucleic acid.
18. The genome editing system of claim 17 , wherein the rate of off-target editing by the genome editing system is about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% less than the off-target editing of the control genome editing system.
19. The genome editing system of claim 17 , wherein the rate of off-target editing is measured by assessing a level of indels at the off-target sequence.
20. The genome editing system of claim 15 , comprising a ribonucleoprotein (RNP) complex comprising the polypeptide and the guide nucleic acid.