Protected guide RNAS (pgRNAS)
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, wherein the guide sequence is modified by secondary structure to increase the specificity of the CRISPR-Cas system and whereby the secondary structure can protect against exonuclease activity and allow for 5′ additions to the guide sequence.
1. A method of modifying a genomic locus of interest in a eukaryotic cell, comprising introducing into the eukaryotic cell an engineered composition comprising or encoding:
(a) a Cas9 protein;
(b) a chimeric RNA comprising a CRISPR-Cas system RNA linked to a protector RNA; wherein the CRISPR-Cas system RNA comprises, from 5′ to 3′, a guide sequence capable of hybridizing to a target sequence at the genomic locus of interest and directing sequence-specific binding of a CRISPR complex to the target sequence, a tracr-mate sequence, and a tracr sequence capable of hybridizing to the tracr-mate sequence; and wherein the protector RNA comprises at least 8 contiguous nucleotides that are complementary to the guide sequence.
2. The method of claim 1 , wherein the composition comprises a vector encoding the chimeric RNA and the Cas9.
3. The method of claim 2 , wherein the vector is a viral vector.
4. The method of claim 3 , wherein the viral vector is an AAV vector.
5. The method of claim 1 , wherein the composition comprises the chimeric RNA and the Cas9.
6. The method of claim 1 , wherein the composition comprises the chimeric RNA and an mRNA encoding the Cas9.
7. The method of claim 1 , wherein the Cas9 comprises one or more mutations in a catalytic domain.
8. The method of claim 7 , wherein the Cas9 comprises one or more mutations in a RuvC domain selected from the group consisting of D10A, E762A and D986A.
9. The method of claim 7 , wherein the Cas9 comprises a D10A mutation.
10. The method of claim 7 , wherein the Cas9 comprises one or more mutations in a HNH domain selected from the group consisting of H840A, N854A and N863A.
11. The method of claim 7 , wherein the Cas9 comprises a H840A mutation.
12. The method of claim 1 , wherein the Cas9 is S. pyogenes Cas9.
13. The method of claim 1 , wherein the Cas9 is S. aureus Cas9.
14. The method of claim 1 , wherein the Cas9 comprises at least one nuclear localization sequence (NLS).
15. The method of claim 1 , wherein the Cas9 comprises at least two NLSs.
16. The method of claim 1 , wherein the Cas9 is fused to a heterologous functional protein domain.
17. The method of claim 1 , wherein the guide sequence comprises 10 to 30 nucleotides in length.
18. The method of claim 1 , wherein the protector RNA comprises at least 10 contiguous nucleotides that are complementary to the guide sequence.
19. The method of claim 1 , wherein the protector RNA comprises at least 12 contiguous nucleotides that are complementary to the guide sequence.
20. The method of claim 1 , wherein the protector RNA comprises 8 to 18 contiguous nucleotides that are complementary to the guide sequence.
21. The method of claim 1 , wherein the protector RNA comprises 10 to 16 contiguous nucleotides that are complementary to the guide sequence.
22. The method of claim 1 , wherein the chimeric RNA further comprises an extension sequence that links the protector RNA to the CRISPR-Cas system RNA.
23. The method of claim 1 , wherein the genomic locus of interest encodes a gene product.
24. The method of claim 23 , wherein the gene product is a protein.
25. The method of claim 23 , wherein expression of the gene product is decreased or increased, or activity or function of the gene product is altered.
26. The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.
27. The method of claim 1 , wherein the eukaryotic cell is a human cell.
28. The method of claim 1 , wherein the eukaryotic cell is a plant cell.