Optimized CRISPR-Cas double nickase systems, methods and compositions for sequence manipulation
The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.
1. A method of modifying a genomic locus of interest including minimizing off-target modifications, comprising introducing into a eukaryotic cell containing a double stranded DNA molecule, an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas9 protein having one or more mutations wherein the one or more mutations is in a RuvC or HNH domain and two guide RNAs that target a first strand and a second strand of the DNA molecule respectively, whereby the Cas9 protein nicks each of the first strand and the second strand of the DNA molecule; wherein the Cas9 protein and the two guide RNAs do not naturally occur together; wherein the Cas9 protein nicking each of the first strand and the second strand of the DNA molecule results in at least one 5′ overhang of at least 26 nucleotides.
2. The method of claim 1 , wherein the DNA molecule encodes a gene product.
3. The method of claim 1 , wherein the 5′ overhang is at most 100 nucleotides.
4. The method of claim 1 , wherein the 5′ overhang is at most 50 nucleotides.
5. The method of claim 1 , wherein the 5′ overhang is at least 30 nucleotides.
6. The method of claim 1 , wherein the 5′ overhang is 34-50 nucleotides.
7. A method of modifying a genomic locus of interest comprising introducing into a eukaryotic cell containing a double stranded DNA molecule, an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas9 protein having one or more mutations wherein the one or more mutations is in a RuvC or HNH domain and four guide RNAs that target a first locus and a second locus of a first strand and a first locus and a second locus of a second strand of the DNA molecule respectively, whereby the Cas9 protein nicks each of the first and second loci of the first strand and the first and second loci of the second strand of the DNA molecule; wherein the Cas9 protein and the four guide RNAs do not naturally occur together; wherein the Cas9 protein nicking each of the first and second loci of the first strand and the first and second loci of the second strand of the DNA molecule results in the double stranded DNA molecule having at least one 5′ overhang of at least 26 nucleotides.
8. The method of claim 7 , wherein the 5′ overhang is at most 100 nucleotides.
9. The method of claim 7 , wherein the 5′ overhang is at most 50 nucleotides.
10. The method of claim 1 or 7 , wherein the guide RNAs comprise a guide sequence fused to a tracr mate sequence and a tracr sequence.
11. The method of claim 1 or 7 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
12. The method of claim 11 , wherein the eukaryotic cell is a mammalian cell.
13. The method of claim 12 , wherein the mammalian cell is a human cell.
14. The method of claim 7 , wherein the DNA molecule encodes a gene product.
15. The method of claim 14 , wherein the Cas9 protein is from or is derived from a genus belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter.
16. The method of claim 15 , wherein the Cas9 protein comprises one or more mutations in a catalytic domain.
17. The method of claim 16 , wherein the one or more mutations is in a RuvC domain.
18. The method of claim 17 , wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.
19. The method of claim 18 , wherein the Cas9 protein has the D10A mutation.
20. The method of claim 16 , wherein the one or more mutations is in a HNH domain.
21. The method of claim 20 , wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.
22. The method of claim 21 , wherein the Cas9 protein has the H840A mutation.
23. The method of claim 2 or 14 , wherein expression of the gene product is decreased.
24. The method of claim 1 or 7 , wherein a template polynucleotide is further introduced into the DNA molecule.
25. The method of claim 2 or 14 , wherein expression of the gene product is increased or activity or function of the gene product is altered.
26. The method of claim 2 or 14 , wherein the gene product is a protein.
27. The method of claim 24 , wherein the template polynucleotide is a double strand or a single strand polynucleotide sequence.
28. The method of claim 24 , wherein the template polynucleotide is introduced into the DNA molecule by homologous recombination.
29. The method of claim 24 , wherein the template polynucleotide comprises a restriction endonuclease restriction site.
30. The method of claim 7 , wherein an intervening sequence is excised allowing 5′ overhangs to reanneal and ligate.
31. A method of modifying a genomic locus of interest, comprising
introducing into a eukaryotic cell containing a double stranded DNA molecule, an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas9 protein having a D 10A mutation and two guide RNAs that target a first strand and a second strand of the DNA molecule respectively, whereby the Cas9 protein nicks each of the first strand and the second strand of the DNA molecule, and a repair template polynucleotide;
wherein the Cas9 protein and the two guide RNAs do not naturally occur together; wherein the Cas9 protein nicking each of the first strand and the second strand of the DNA molecule results in at least one 5′ overhang, and wherein the 5′ overhang is 26-100 nucleotides and facilitates homology-directed repair by the repair template polynucleotide.
32. The method of claim 1 , wherein the Cas9 is a S. pyogenes Cas9.
33. The method of claim 1 , wherein the Cas9 is a S. aureus Cas9.
34. The method of claim 1 , wherein the Cas9 comprises at least one nuclear localization sequence (NLS).
35. The method of claim 1 , wherein the Cas9 comprises at least two NLSs.
36. The method of claim 1 , wherein the Cas9 comprises at least one N-terminal NLS and at least one C-terminal NLS.