Crispr enzyme mutations reducing off-target effects
Disclosed and claimed are mutation(s) or modification(s) of the CRISPR enzyme, for example a Cas enzyme such as a Cas9, which obtain an improvement, for instance a reduction, as to off-target effects of a CRISPR-Cas or CRISPR-enzyme or CRISPR-Cas9 system or complex containing or including such a mutated or modified Cas or CRISPR enzyme or Cas9. Methods for making and using and uses of such mutated or modified Cas or CRISPR enzyme or Cas9 and systems or complexes containing the same and products from such methods and uses are also disclosed and claimed.
1. An engineered Cas9 protein comprising an HNH domain, a RuvC domain, and two or more modifications in a groove between the HNH and RuvC domains, wherein each of the modifications comprises an amino acid substitution of a positively charged residue of wild-type Cas9 with an uncharged residue, wherein the modifications are amino acid substitutions at positions selected from the group consisting of 780, 810, 848, 855, 976, 982, 1003, and 1060, with reference to amino acid position numbering of S. pyogenes Cas9 (SpCas9).
2. The engineered Cas9 protein of claim 1 , wherein the uncharged residue is alanine.
3. The engineered Cas9 protein of claim 1 , wherein the modifications alter binding kinetics at DNA:RNA:protein.
4. The engineered Cas9 protein of claim 1 , wherein the modifications comprise amino acid substitutions, with reference to numbering of SpCas9: R780A and K810A, or R780A and K855A, or R780A and R976A, or K848A and R976A, or K855A and R976A, or R780A and K848A, or K810A and K848A, or K848A and K855A, or K810A and K855A, or H982A and R1060A, or H982A and R1003A, or K1003A and R1060A, or R780A and H982A, or K810A and H982A, or K848A and H982A, or K855A and H982A, or R780A and K1003A, or K810A and R1003A, or K848A and K1003A, or R780A and R1060A, or K810A and R1060A, or K848A and R1060A, or K848A and EI 108A, or K810A and K1003A, or R780A and R1060A, or K810A and R1060A, or K848A and R1060A.
5. The engineered Cas9 protein of claim 1 , wherein modifications comprise amino acid substitutions, with reference to numbering of SpCas9: H982A, K1003A, and K1 129E; or R780A, K1003A, and R1060A; or K810A, K1003A, and R1060A; or K848A, K1003A, and R1060A; or K855A, K1003A, and R1060A; or H982A, K1003A, and R1060A.
6. The engineered Cas9 protein of claim 1 , wherein the modifications comprise, with reference to the amino acid numbering of SpCas9: K848A, K1003A, and R1060A.
7. The engineered Cas9 protein of claim 1 , wherein the protein is a mutant of a Cas9 protein from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium and Corynebacter.
8. The engineered Cas9 protein of claim 1 , wherein the protein comprises one or more nuclear localization signal (NLS).
9. The engineered Cas9 protein of claim 1 , wherein the protein comprises at least two or more NLSs.
10. The engineered Cas9 protein of claim 1 , wherein the protein comprises three or more modifications.
11. A CRISPR-Cas complex, comprising the engineered Cas9 protein of claim 1 and a guide polynucleotide.
12. The CRISPR-Cas complex of claim 11 , wherein the guide polynucleotide comprises a guide sequence, a tracr mate sequence, and a tracr sequence.
13. The CRISPR-Cas complex of claim 12 , wherein the guide polynucleotide is a chimeric guide polynucleotide in which the tracr mate sequence is linked to the tracr sequence.
14. A method of modulating gene expression or modifying a target sequence in a cell or cell line, comprising introducing the CRISPR-Cas complex of claim 11 into the cell or cell line, whereby the gene expression is modulated or the target sequence is modified resulting in a modified cell or cell line.
15. The method of claim 14 , further comprising culturing the modified cell or cell to produce progeny.
16. The method of claim 14 , wherein the cell or cell line is a eukaryotic cell or cell line.
17. The method of claim 14 , wherein the CRISPR-Cas complex is introduced into the cell or cell line ex vivo.
18. A method of producing a plant having a modified trait of interest encoded by a gene of interest, comprising delivery to a plant cell of the CRISPR-Cas complex of claim 11 , whereby the gene of interest is either modified or introduced into the cell and a modified plant cell is obtained, and regenerating the plant from the modified plant cell.
19. The method of claim 18 , further comprising obtaining seed from the plant.
20. A method of modifying a non-human organism by manipulation of one or more target sequences, comprising delivering to the organism the CRISPR-Cas complex of claim 11 , thereby obtaining a modified non-human organism.
21. A method according to claim 20 , wherein the non-human organism is a plant or algae.
22. A method according to claim 20 , wherein the non-human organism is an animal.