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. An engineered, non-naturally occurring composition comprising a CRISPR Cas system comprising:
(I) a Cas9 protein or a polynucleotide encoding the Cas9 protein; and
(II) a protected guide or a polynucleotide encoding the protected guide, wherein the protected guide comprising, from 5′ to 3′, (a) a protector sequence, (b) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of a CRISPR complex to the target sequence, (c) a tracr mate sequence, and (d) a tracr sequence capable of hybridizing to the tracr mate sequence, wherein the protector sequence comprises three or more nucleotides that are complementary to the guide sequence and two or more nucleotides that are non-complementary to the target sequence, and wherein the protected guide comprises a hairpin formed by hybridization between the protector sequence and the guide sequence.
2. The composition of claim 1 , wherein the protected guide is a chimeric RNA.
3. The composition of claim 1 , wherein the protector sequence has a length between 3 and 120 nucleotides.
4. The composition of claim 1 , wherein the guide sequence comprises a protected part and an exposed part, and the exposed part is 1 to 19 nucleotides.
5. The composition of claim 1 , wherein the guide sequence is at least 90% or about 100% complementary to the protector sequence.
6. The composition of claim 1 , wherein the protected guide further comprises an extension sequence between the 5′ end of the guide sequence and the 3′ end of the protector sequence.
7. The composition of claim 6 , wherein the extension sequence is 100% not complementary to the protector sequence.
8. The composition of claim 1 , wherein the guide sequence further comprises mismatches appended to the end of the guide sequence, and wherein the mismatches thermodynamically optimize specificity.
9. The composition of claim 1 , wherein the Cas9 comprises one or more nuclear localization sequences.
10. An isolated cell comprising the composition of claim 9 .
11. The isolated cell of claim 10 , wherein the cell is a eukaryotic cell.
12. The isolated cell of claim 11 , wherein the eukaryotic cell is a mammalian cell.
13. The isolated cell of claim 12 , wherein the mammalian cell is a human cell.
14. The composition of claim 1 , wherein the protector sequence is 10-30 nucleotides.
15. The composition of claim 1 , wherein the guide sequence is 10-30 nucleotides.
16. The composition of claim 1 , wherein the guide sequence is at least 75% complementary to the target sequence.
17. The composition of claim 1 , wherein the guide sequence is at least 90% complementary to the target sequence.
18. The composition of claim 1 , wherein the guide sequence is about 100% complementary to the target sequence.
19. The composition of claim 1 , wherein the tracr mate sequence is at least 75% complementary to the tracr sequence.
20. The composition of claim 1 , wherein the tracr mate sequence is at least 90% complementary to the tracr sequence.
21. The composition of claim 1 , wherein the tracr mate sequence is about 100% complementary to the tracr sequence.
22. The composition of claim 4 , wherein the exposed part of the guide sequence is at least 75% complementary to the target sequence.
23. The composition of claim 4 , wherein the exposed part of the guide sequence is at least 90% complementary to the target sequence.
24. The composition of claim 6 , wherein the extension sequence is 12 nucleotides or less.
25. The composition of claim 6 , wherein the extension sequence is at least 70% not complementary to the protector sequence.
26. The composition of claim 6 , wherein the extension sequence is at least 80% not complementary to the protector sequence.
27. The composition of claim 6 , wherein the extension sequence is at least 90% not complementary to the protector sequence.
28. The composition of claim 9 , wherein the Cas9 comprises at least one mutation in the RuvC and/or HNH domain and has no more than 5% of the nuclease activity of a corresponding wild-type Cas9.
29. The composition of claim 9 , wherein the Cas9 comprises at least two mutations in the RuvC and/or HNH domain and has diminished nuclease activity of at least 97% or 100% as compared with a corresponding wild-type Cas9.