CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
View Patent ↗The invention provides for 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. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.
1. A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence 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 Type-II Cas9 protein, 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 whereby the Cas9 protein is a nickase that cleaves only one strand of the DNA molecule, whereby the guide RNA targets the target sequence and the Cas9 protein nicks the DNA molecule, 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 method further comprises the insertion of a recombination template into the nicked DNA molecule.
3. The method of claim 1 , wherein the expression of two or more gene products is altered.
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 Cas9 protein comprises a mutation, wherein the mutation comprises D10A, E762A, H840A, N854A, N863A or D986A with reference to the position numbering of a Streptococcus pyogenes Cas9 protein.
6. The method of claim 1 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
7. The method of claim 1 , wherein the eukaryotic cell is a mammalian or human cell.
8. The method of claim 1 , wherein the expression of one or more gene products is altered by genome editing.
9. The method of claim 1 , wherein the expression of one or more gene products is increased.
10. The method of claim 1 , wherein the expression of one or more gene products is decreased.
11. The method of claim 1 , wherein the one or more vectors are viral vectors.
12. The method of claim 1 , wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
13. A CRISPR-Cas system-mediated genome editing method comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence 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 Type-II Cas9 protein, 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 whereby the Cas9 protein is a nickase that cleaves only one strand of the DNA molecule, whereby 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 CRISP R-Cas system, whereby there is genome editing; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.
14. The method of claim 13 , wherein the method further comprises the insertion of a recombination template into the nicked DNA molecule.
15. The method of claim 13 , wherein the expression of two or more gene products is altered.
16. The method of claim 13 , wherein the CRISPR-Cas system comprises a tracr sequence.
17. The method of claim 13 , wherein the Cas9 protein comprises a mutation, wherein the mutation comprises D10A, E762A, H840A, N854A, N863A or D986A with reference to the position numbering of a Streptococcus pyogenes Cas9 protein.
18. The method of claim 13 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
19. The method of claim 13 , wherein the eukaryotic cell is a mammalian or human cell.
20. The method of claim 13 , wherein the expression of one or more gene products is increased.
21. The method of claim 13 , wherein the expression of one or more gene products is decreased.
22. The method of claim 13 , wherein the one or more vectors are viral vectors.
23. The method of claim 13 , wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
24. An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprising a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product and wherein the Cas9 protein comprises one or more mutations in a catalytic domain whereby the Cas9 protein is a nickase that cleaves only one strand of the DNA molecule, 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.
25. The CRISPR-Cas system of claim 24 , wherein the CRISPR-Cas system comprises a tracr sequence.
26. The CRISPR-Cas system of claim 24 , wherein the Cas9 protein comprises a mutation, wherein the mutation comprises D10A, E762A, H840A, N854A, N863A or D986A with reference to the position numbering of a Streptococcus pyogenes Cas9 protein.
27. The CRISPR-Cas system of claim 26 , wherein the Cas9 protein comprises the mutation D10A.
28. The CRISPR-Cas system of claim 26 , wherein the Cas9 protein comprises the mutation H840A.
29. The CRISPR-Cas system of claim 24 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
30. The CRISP R-Cas system of claim 24 , wherein the eukaryotic cell is a mammalian or human cell.