Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
View Patent ↗The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are compositions and methods related to components of a CRISPR complex particularly comprising a Cas ortholog enzyme.
1. A modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) enzyme wherein the enzyme is a Cas9 protein that is a chimeric CRISPR enzyme in that it comprises a first fragment and a second fragment, wherein each of the first and second fragments is from a different Cas9 protein from a bacteria 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 , and Campylobacter.
2. The modified CRISPR enzyme of claim 1 wherein the C terminus of the modified CRISPR enzyme is from a Streptococcus pyogenes (Spy Cas9 enzyme.
3. The modified CRISPR enzyme of claim 1 wherein the N terminus of the modified CRISPR enzyme is from a Sp Cas9 enzyme.
4. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC I domain of a Sp Cas9 enzyme.
5. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC II domain of a Sp Cas9 enzyme.
6. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC III domain of a Sp Cas9 enzyme.
7. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a HNH domain of a Sp Cas9 enzyme.
8. The modified CRISPR enzyme of claim 1 wherein the N terminus of the modified CRISPR enzyme is from a Streptococcus thermophilus (St) Cas9 enzyme.
9. The modified CRISPR enzyme of claim 1 wherein the C terminus of the modified CRISPR enzyme is from a St Cas9 enzyme.
10. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC I domain of a St Cas9 enzyme.
11. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC II domain of a Sp Cas9 enzyme.
12. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC III domain of a St Cas9 enzyme.
13. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a HNH domain of a St Cas9 enzyme.
14. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′-NGG, wherein N is any nucleotide.
15. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′-NRG, wherein N is any nucleotide and R is a purine.
16. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′-NNGRR, wherein N is any nucleotide and R is a purine.
17. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises a PAM sequence set forth in FIG. 7 or FIG. 13A-II .
18. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′-GAAA, 5′-GAAC, 5′-GAAG, 5′-GAAT, 5′-GAGA, 5′-GAGC, 5′-GAGG, or 5′-GGAA.
19. The modified CRISPR enzyme of claim 1 wherein the chimera comprises a Sp_St3 chimera.
20. The modified CRISPR enzyme of claim 1 wherein the chimera comprises a St3_Sp chimera.
21. A nucleic acid molecule encoding the modified CRISPR enzyme of claim 1 , wherein the nucleic acid molecule is codon-optimized for expression in a eukaryotic cell.
22. A vector comprising: a nucleic acid molecule encoding the modified CRISPR enzyme of claim 1 and further comprising a nucleic acid molecule(s) encoding one or more nuclear localization signal(s) (NLS(s)); and a regulatory element operable in a eukaryotic cell operably linked to the nucleic acid molecules encoding the modified CRISPR enzyme and the NLS(s).
23. An engineered, programmable, non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system that alters expression of at least one gene product in a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding said gene product, wherein the CRISPR-Cas system comprises the modified CRISPR enzyme of claim 1 .
24. The CRISPR-Cas system of claim 23 wherein when introduced into a cell having a DNA molecule, the CRISPR-Cas system cleaves the DNA, and the CRISPR-Cas system includes DNA for insertion into a cleaved strand of the DNA molecule.
25. A method of altering expression of at least one gene product in a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding said gene product comprising introducing into said eukaryotic cell an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and
b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding the modified CRISPR enzyme of claim 1 ,
wherein said CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system;
whereby said guide RNA targets the target sequence and said CRISPR enzyme cleaves the DNA molecule;
whereby expression of said at least one gene product is altered; and, wherein said modified CRISPR enzyme and said guide RNA do not naturally occur together.
26. The method of claim 25 , further comprising inserting DNA into a cleaved strand of the DNA molecule.
27. A CRISP R-Cas system-mediated genome targeting method in a eukaryotic cell containing a DNA molecule having a target sequence comprising introducing into said eukaryotic cell an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and
b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding the modified CRISPR enzyme of claim 1 ,
wherein said CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system;
the method further comprising inserting DNA into a cleaved strand of the DNA molecule;
whereby there is CRISPR-Cas system-mediated genome targeting through said CRISPR-Cas system acting as to the DNA molecule comprising said guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby there is genome editing; and, wherein said modified CRISPR enzyme and said guide RNA do not naturally occur together.
28. The method of claim 27 , wherein the eukaryotic cell is a mammalian or human cell.