Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for targeting disorders and diseases using viral components
The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also 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 to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.
1. An AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV1/2, or AAV2/8 viral vector that contains heterologous nucleic acid molecule(s) engineered for expression in a eukaryotic cell, wherein the heterologous nucleic acid molecule(s) encodes a Cas9 and a CRISPR-Cas system guide, wherein the Cas9 and the system guide are capable of forming a CRISPR-Cas complex after expression in the eukaryotic cell, wherein the system guide is capable of targeting the complex to a DNA sequence in the genome of the eukaryotic cell,
wherein the Cas9 and the system guide are encoded in a single AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV1/2, or AAV2/8 viral vector, and wherein the Cas9 is Staphylococcus aureus Cas9 (SaCas9).
2. The viral vector of claim 1 , wherein one or more of the system guides comprises
(a) a guide sequence that hybridizes to the target sequence,
(b) a tracr mate sequence, and
(c) a tracr sequence,
wherein (a), (b), and (c) are arranged in a 5′ to 3′ orientation.
3. The viral vector of claim 2 , wherein expression of the guide sequence is under the control of a T7 promoter and is driven by the expression of T7 polymerase.
4. The viral vector of claim 1 , wherein the viral vector encodes two or more system guides.
5. The viral vector of claim 1 , wherein one or more of the system guides comprises a chimeric RNA (chiRNA).
6. The viral vector of claim 1 , wherein the heterologous nucleic acid molecule further encodes a homologous recombination (FIR) template.
7. The viral vector of claim 1 , wherein the Cas9 is a nuclease directing cleavage of both strands of the targeted DNA sequence.
8. The viral vector of claim 1 , wherein the Cas9 comprises one or more mutations.
9. The viral vector of claim 8 , wherein the Cas9 comprises one or more of D10A, E762A, H840A, N854A, N863A and D986A.
10. The viral vector of claim 8 , wherein the one or more mutations is in a RuvC1 domain of the Cas9.
11. The viral vector of claim 8 , wherein the Cas9 is a nickase directing cleavage of one strand of the targeted DNA sequence.
12. The viral vector of claim 11 , wherein the nickase is a double nickase.
13. The viral vector of claim 1 , wherein the nucleic acid molecule encoding the Cas9 comprises at least one or more NLS.
14. The viral vector of claim 1 , wherein the nucleic acid molecule encoding the Cas9 comprises at least two or more NLS.
15. The viral vector of claim 1 , wherein the Cas9 has one or more mutations in a catalytic domain, wherein the Cas9 further comprises a functional domain.
16. The viral vector of claim 15 , wherein the functional domain is a transcriptional activation domain.
17. The viral vector of claim 15 , wherein the functional domain is VP64.
18. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of a neural cell.
19. The viral vector of claim 18 , wherein the targeted DNA sequence is associated with a neurological or neuronal disease or disorder in Table 7, 8, or 9.
20. The viral vector of claim 18 , wherein the serotype of the viral vector is AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 or a combination thereof.
21. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of a muscle cell.
22. The viral vector of claim 21 , wherein the muscle cell is a cardiac muscle cell.
23. The viral vector of claim 21 , wherein the targeted DNA sequence is associated with a muscular/skeletal disease or disorder in Table 7, 8, or 9.
24. The viral vector of claim 21 , wherein the condition is Duchenne Muscular Dystrophy (DIV)).
25. The viral vector of claim 21 , wherein the serotype of the viral vector is AAV1, AAV6, AAV7, AAV8, or AAV9.
26. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of a lung cell.
27. The viral vector of claim 26 , wherein the targeted DNA sequence is associated with a lung cell gene or disorder in Table 7, 8, or 9.
28. The viral vector of claim 26 , wherein the condition is cystic fibrosis or alpha-1 antitrypsin deficiency (AAD).
29. The viral vector of claim 26 , wherein the serotype of the viral vector is AAV1, AAV2, AAV5, AAV6, or AAV9.
30. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of a hepatic cell.
31. The viral vector of claim 30 , wherein the targeted DNA sequence is associated with a hepatic cell gene or disorder in Table 7, 8, or 9.
32. The viral vector of claim 30 , wherein the condition is a glycogen storage disease or alpha-1 antitrypsin deficiency (AAD).
33. The viral vector of claim 30 , wherein the serotype of the viral vector is AAV1, AAV5, AAV7, AAV8, AAV2/8, or AAV9.
34. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of a pancreatic cell.
35. The viral vector of claim 34 , wherein the targeted DNA sequence is associated with a pancreatic cell gene or disorder in Table 7, 8, or 9.
36. The viral vector of claim 34 , wherein the serotype of the viral vector is AAV6 or AAV8.
37. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of an epithelial cell.
38. The viral vector of claim 37 , wherein the targeted DNA sequence is associated with an epithelial gene or disorder in Table 7, 8, or 9.
39. The viral vector of claim 37 , wherein the serotype of the viral vector is AAV1, AAV2, AAV5, AAV6, or AAV9.
40. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a condition or defect of a cell of the eye.
41. The viral vector of claim 40 , wherein the targeted DNA sequence is associated with an ocular gene or disorder in Table 7, 8, or 9.
42. The viral vector of claim 40 , wherein the ocular condition is Leber's Congenital Amaurosis (LCA) or Usher Syndrome.
43. The viral vector of claim 40 , wherein the serotype of the viral vector is AAV1, AAV2, AAV4, AAV5, or AAV8.
44. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with an immune-related disease or disorder.
45. The viral vector of claim 44 , wherein the targeted DNA sequence is associated with an immune-related gene or disorder in Table 7, 8, or 9.
46. The viral vector of claim 44 , wherein the immune-related disease or disorder is severe combined immunodeficiency (SCID).
47. The viral vector of claim 1 , wherein the targeted DNA sequence is associated with a blood disease or disorder.
48. The viral vector of claim 47 , wherein the targeted DNA sequence is associated with a blood gene or disorder in Table 7, 8, or 9.
49. The viral vector of claim 47 , wherein the blood disease or disorder is hemophilia, sickle cell disease, or beta-thalassemia.
50. An isolated cell comprising a viral vector of claim 1 .
51. A non-human organism comprising a cell of claim 50 .
52. The non-human organism of claim 51 , wherein the organism is an eukaryote.
53. The non-human organism of claim 51 , wherein the organism is a mammal.
54. An isolated tissue comprising the cell of claim 50 .
55. An non-human organism comprising the tissue of claim 54 .