Mutations in
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 increased 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 increased on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system.
1. 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 increased on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system having a wild-type Cas9 protein of SEQ ID NO:1, wherein the isolated mutant Cas9 protein comprises the double amino acid substitution D54A/R691A introduced into SEQ ID NO:1.
2. An isolated ribonucleoprotein complex, comprising:
a mutant Cas9 protein; 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 increased on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system having a wild-type Cas9 protein of SEQ ID NO:1, wherein the mutant Cas9 protein comprises the double amino acid substitution D54A/R691A introduced into SEQ ID NO:1.
3. The isolated ribonucleoprotein complex of claim 2 , wherein the gRNA comprises a crRNA and a tracrRNA in stoichiometric ratio.
4. The isolated ribonucleoprotein complex of claim 3 , wherein the gRNA comprises:
an isolated crRNA comprises an isolated, synthetic crRNA directed against a specific editing target site for a given locus; and
the tracrRNA comprises an isolated, synthetic tracrRNA.
5. The isolated ribonucleoprotein complex of claim 3 , wherein the gRNA comprises a sgRNA.
6. An isolated nucleic acid encoding a mutant Cas9 protein, wherein the mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays increased on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system having a wild-type Cas9 protein of SEQ ID NO: 1, wherein the mutant Cas9 protein comprises the double amino acid substitution D54A/R691A introduced into SEQ ID NO:1.
7. A CRISPR/Cas endonuclease system comprising a mutant Cas9 protein and a gRNA, wherein the CRISPR/Cas endonuclease system displays increased on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system having a wild-type Cas9 protein of SEQ ID NO:1, wherein the mutant Cas9 protein comprises the double amino acid substitution D54A/R691A introduced into SEQ ID NO:1.
8. The CRISPR/Cas endonuclease system of claim 7 , wherein the CRISPR/Cas endonuclease system further comprises a DNA expression vector, wherein the mutant Cas9 protein is encoded by the DNA expression vector.
9. The CRISPR/Cas endonuclease system of claim 8 , wherein the DNA expression vector comprises a plasmid-borne vector.
10. The CRISPR/Cas endonuclease system of claim 8 , wherein the DNA expression vector is selected from a bacterial expression vector and a eukaryotic expression vector.
11. The CRISPR/Cas endonuclease system of claim 7 , wherein the gRNA includes a crRNA and a tracrRNA in stoichiometric ratio.
12. The CRISPR/Cas endonuclease system of claim 11 , wherein the crRNA comprises an isolated, synthetic crRNA directed against a specific editing target site for a given locus and the tracrRNA comprises an isolated, synthetic tracrRNA.
13. The CRISPR/Cas endonuclease system of claim 7 , wherein the gRNA comprises a sgRNA.
14. A method of performing gene editing having 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 increased on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system having a wild-type Cas9 protein of SEQ ID NO:1, wherein the mutant Cas9 protein comprises the double amino acid substitution D54A/R691A introduced into SEQ ID NO:1.