Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
The invention provides for 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 with additional functional domains. 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 altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product 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 a fusion of a Type-II Cas9 protein and one or more protein domains,
wherein:
components (a) and (b) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain,
the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length,
whereby expression of the at least one gene product is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.
2. The method of claim 1 , wherein the CRISPR-Cas system further comprises a flexible linker between the Cas9 protein and the one or more protein domains.
3. The method of claim 1 , wherein the one or more protein domains comprises one or more nuclear localization signal(s) (NLS(s)).
4. The method of claim 1 , wherein the CRISPR-Cas system comprises a trans-activating cr (tracr) sequence.
5. The method of claim 1 , wherein the guide RNA comprises a guide sequence and a tracr sequence.
6. The method of claim 1 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell and/or the eukaryotic cell is a mammalian and/or human cell.
7. The method of claim 1 , wherein the one or more protein domains comprises one or more activator(s).
8. The method of claim 1 , wherein the expression of one or more gene products is increased.
9. The method of claim 1 , wherein the expression of one or more gene products is decreased.
10. The method of claim 1 , wherein the one or more vectors are viral vectors.
11. The method of claim 1 , wherein the viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
12. A CRISPR-Cas system-mediated genome targeting method comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding at least one gene product 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 a fusion of a Type-II Cas9 protein and one or more protein domains,
wherein components (a) and (b) are located on same or different vectors of the system, wherein the Cas9 protein comprises one or more mutations in a catalytic domain,
wherein the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length,
whereby expression of the at least one gene product is altered through the CRISP R-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition, whereby there is genome targeting; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.
13. The method of claim 12 , wherein the CRISPR-Cas system further comprises a flexible linker between the Cas9 protein and the one or more protein domains.
14. The method of claim 12 , wherein the one or more protein domains comprises one or more NLS(s).
15. The method of claim 12 , wherein the CRISPR-Cas system comprises a tracr sequence.
16. The method of claim 12 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell and/or the eukaryotic cell is a mammalian or human cell.
17. The method of claim 12 , wherein the one or more protein domains comprises one or more activator(s).
18. The method of claim 12 , wherein the expression of one or more gene products is increased.
19. The method of claim 12 , wherein the expression of one or more gene products is decreased.
20. The method of claim 12 , wherein the one or more vectors are viral vectors.
21. The method of claim 12 , wherein the viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
22. An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprising a fusion of a Cas9 protein and one or more protein domains, and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell, wherein the DNA molecule encodes at least one gene product, wherein the CRISPR-Cas system comprises a tracr sequence, wherein the Cas9 protein comprises one or more mutations in a catalytic domain, wherein the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, whereby when the CRISPR-Cas system is introduced into the eukaryotic cell expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.
23. The CRISPR-Cas system of claim 22 , wherein the CRISP R-Cas system further comprises a flexible linker between the Cas9 protein and the one or more protein domains.
24. The CRISPR-Cas system of claim 22 , wherein the one or more protein domains comprises one or more NLS(s): or activator(s); or repressor(s).
25. The CRISPR-Cas system of claim 22 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell and/or the eukaryotic cell is a mammalian or human cell.
26. The CRISPR-Cas system of claim 22 , wherein the one or more protein domains comprises one or more activator(s).
27. The CRISPR-Cas system of claim 22 , wherein the expression of one or more gene products is increased.
28. The CRISP R-Cas system of claim 22 , wherein the expression of one or more gene products is decreased.