CRISPR-Cas systems and methods for altering expression of gene products
View Patent ↗The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. 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 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 viral 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,
whereby the guide RNA targets the target sequence and the Cas9 protein cleaves 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 expression of two or more gene products is altered.
3. The method of claim 1 , wherein the CRISPR-Cas system further 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 RNAs comprise a guide sequence fused to a tracr sequence.
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 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 viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
11. 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,
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 CRISPR-Cas system, whereby there is genome editing; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.
12. The method of claim 11 , wherein the expression of two or more gene products is altered.
13. The method of claim 11 , wherein the CRISPR-Cas system further comprises one or more NLS(s).
14. The method of claim 11 , wherein the CRISPR-Cas system comprises a tracr sequence.
15. The method of claim 11 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
16. The method of claim 11 , wherein the eukaryotic cell is a mammalian or human cell.
17. The method of claim 11 , wherein the expression of one or more gene products is increased.
18. The method of claim 11 , wherein the expression of one or more gene products is decreased.
19. An engineered, non-naturally occurring CRISPR-Cas system comprising one or more viral 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 a target sequence of a DNA molecule in a eukaryotic cell that contains the DNA molecule, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product,
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 CRISPR-Cas system comprises a tracr sequence,
whereby the guide RNA targets and hybridizes with the target sequence and the Cas9 protein cleaves 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.
20. The system of claim 19 , wherein the CRISPR-Cas system further comprises one or more NLS(s).
21. The system of claim 19 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
22. The system of claim 19 , wherein the eukaryotic cell is a mammalian or human cell.
23. The system of claim 19 , wherein the expression of one or more gene products is increased.
24. The system of claim 19 , wherein the expression of one or more gene products is decreased.
25. The system of claim 19 , wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.
26. 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 the Cas9 protein cleaves the DNA molecule and wherein the CRISP R-Cas system comprises a tracr sequence, 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.
27. The CRISPR-Cas system of claim 26 , wherein the CRISPR-Cas system further comprises one or more NLS(s).
28. The CRISPR-Cas system of claim 26 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
29. The CRISPR-Cas system of claim 26 , wherein the eukaryotic cell is a mammalian or human cell.