Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems. In particular the present invention comprehends engineered new guide architectures and enzymes to be used in optimized Staphylococcus aureus CRISPR-Cas enzyme systems.
1. An engineered SaCas9 protein comprising at least one modification compared to a wild-type SaCas9 protein, wherein the modification comprises a mutation of at least one PAM-interacting residue, wherein the PAM-interacting residue is Y789, Y882, K886, N888, A889, L909, N985, N986, R991, or E993, and wherein the engineered SaCas9 protein is non-naturally occurring.
2. The engineered SaCas9 protein of claim 1 , further comprising a modification or a mutation of one or more nuclease domains.
3. The engineered SaCas9 protein of claim 2 , wherein the modification or mutation comprises a modification or a mutation of a RuvC domain or an HNH domain.
4. The engineered SaCas9 protein of claim 1 , further comprising an amino acid substitution selected from the group consisting of E477A, N580D, N580A, D1OA, H701 A, D704A, H577A, and a combination thereof.
5. The engineered SaCas9 protein of claim 1 , further comprising an amino acid substitution in a binding groove.
6. The engineered SaCas9 protein of claim 5 , wherein the binding groove is between a RuvC and an HNH domain.
7. The engineered SaCas9 protein of claim 1 , further comprising a fusion of one or more heterologous functional domains.
8. The engineered SaCas9 protein of claim 7 , wherein the heterologous function domain comprises a transcription activation domain, a transcription repressor domain, or a nuclease domain.
9. The engineered SaCas9 protein of claim 7 , wherein the heterologous functional domain is inserted into a linker loop between amino acids 430 and 431.
10. The engineered SaCas9 protein of claim 7 , wherein the heterologous functional domain is inserted in a RuvC-III domain between amino acids 739 and 740.
11. The engineered SaCas9 protein of claim 7 , wherein the heterologous functional domain comprises a VP64 domain, a KRAB domain, a SID domain, or a FokI domain.
12. The engineered SaCas9 protein of claim 7 , wherein the one or more heterologous functional domains comprise one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, and nucleic acid binding activity.
13. The engineered SaCas9 protein of claim 1 , wherein the engineered SaCas9 protein comprises a split SaCas9 dimer structure.
14. The engineered SaCas9 protein of claim 13 , wherein the engineered SaCas9 protein comprises a split in a RuvC domain.
15. The engineered SaCas9 protein of claim 13 , wherein the engineered SaCas9 protein comprises a split between amino acid 739 and amino acid 740.
16. The engineered SaCas9 protein of claim 13 , wherein the engineered SaCas9 protein comprises a split in a linker between a RuvC-II domain and an HNH domain.
17. The engineered SaCas9 protein of claim 13 , wherein the engineered SaCas9 protein comprises a split between amino acid 430 and amino acid 431.
18. The engineered SaCas9 protein of claim 1 , wherein the engineered SaCas9 protein comprises one or more nuclear localization signal (NLS) domains.
19. The engineered SaCas9 protein of claim 1 , wherein the engineered SaCas9 protein comprises at least two or more NLSs.
20. A composition comprising the engineered SaCas9 protein of claim 1 .
21. A system comprising the engineered SaCas9 protein of claim 1 , and a CRISPR-Cas system RNA capable of forming a CRISPR-Cas complex with the engineered SaCas9 protein.
22. The engineered SaCas9 protein of claim 1 , wherein the PAM-interacting residue is N985.
23. The engineered SaCas9 protein of claim 1 , wherein the PAM-interacting residue is N986.
24. The engineered SaCas9 protein of claim 1 , wherein the PAM-interacting residue is R991.
25. The engineered SaCas9 protein of claim 1 , wherein the PAM-interacting residue is E993.
26. A vector system comprising one or more vectors, wherein the one or more vectors comprises: a) a first regulatory element operably linked to a nucleotide sequence encoding the engineered SaCas9 protein of claim 1 ; and b) a second regulatory element operably linked to one or more nucleotide sequences encoding a CRISPR-Cas system RNA capable of forming a CRISPR-Cas complex with the engineered SaCas9 protein, wherein the CRISPR-Cas system RNA comprises a guide sequence, a tracr sequence, and a tracr mate sequence, wherein components (a) and (b) are located on same or different vectors.
27. The vector system of claim 26 , wherein the engineered SaCas9 protein is encoded by a nucleotide sequence which is codon optimized for expression in a eukaryote.