Materials and methods for treatment of autosomal dominant retinitis pigmentosa

1. A self-inactivating CRISPR-Cas system comprising:

a first segment comprising a nucleotide sequence that encodes Cas9;

a second segment comprising a nucleotide sequence that encodes a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA) wherein the gRNA or the sgRNA comprises SEQ ID NO: 5290 or 5291; and

one or more third segments comprising a self-inactivating (SIN) site, wherein the SIN site is:

a 5′ SIN site located upstream of the nucleotide sequence that encodes Cas9 and downstream of a SV40 nuclear localization signal (NLS); or

a 3′ SIN site located within a naturally occurring or chimeric inserted intron located within the nucleotide sequence that encodes Cas9;

wherein the gRNA or sgRNA is complementary to the SIN site;

wherein the gRNA or sgRNA is complementary to a genomic target sequence.

2. The self-inactivating CRISPR-Cas system of claim 1 , wherein the Cas9 is Staphylococcus aureus Cas9 (SaCas9).

3. The self-inactivating CRISPR-Cas system of claim 1 , wherein the 5′ SIN site comprises SEQ ID NO: 5300 or 5301.

4. The self-inactivating CRISPR-Cas system of claim 1 , wherein the 3′ SIN site comprises SEQ ID NO: 5280 or 5281.

5. The self-inactivating CRISPR-Cas system of claim 1 , where the SIN site comprises a protospacer adjacent motif (PAM).

6. The self-inactivating CRISPR-Cas system of claim 5 , wherein the PAM is NNGRRT.

7. The self-inactivating CRISPR-Cas system of claim 1 , wherein the genomic target sequence is a P23H mutation in a rhodopsin (RHO) gene.

8. The self-inactivating CRISPR-Cas system of claim 1 , wherein the first segment comprising a nucleotide sequence that encodes Cas9, further comprises a start codon, a stop codon, and a poly(A) termination site.

9. The self-inactivating CRISPR-Cas system of claim 1 , wherein the first segment and the third segment are provided together in a first vector and the second segment is provided in a second vector.

10. The self-inactivating CRISPR-Cas system of claim 1 , wherein the first segment, second segment, and third segment are provided together in a vector.

11. The self-inactivating CRISPR-Cas system of claim 9 , wherein the first vector comprises SEQ ID NO: 5341 or 5342.

12. The self-inactivating CRISPR-Cas system of claim 9 , wherein the second vector comprises SEQ ID NO: 5339 or 5340.

13. The self-inactivating CRISPR-Cas system of claim 1 , wherein the third segment is less than 100 nucleotides in length.

14. The self-inactivating CRISPR-Cas system of claim 1 , wherein the gRNA or the sgRNA is complementary to the nucleotide sequence of the SIN site except for in at least one location.

15. The self-inactivating CRISPR-Cas system of claim 1 , wherein a nucleic acid sequence encoding a promoter is operably linked to the first segment.

16. The self-inactivating CRISPR-Cas system of claim 15 , wherein the promoter is a spatially-restricted promoter, bidirectional promoter, or an inducible promoter.

17. The self-inactivating CRISPR-Cas system of claim 16 , wherein the spatially-restricted promoter is selected from the group consisting of: any tissue or cell type specific promoter, a hepatocyte-specific promoter, a neuron-specific promoter, an adipocyte-specific promoter, a cardiomyocyte-specific promoter, a skeletal muscle-specific promoter, lung progenitor cell specific promoter, a photoreceptor-specific promoter, and a retinal pigment epithelial (RPE) selective promoter.

18. The self-inactivating CRISPR-Cas system of claim 9 , wherein the first vector and the second vector are adeno-associated virus (AAV) vectors.

19. The self-inactivating CRISPR-Cas system of claim 18 , wherein the AAV vectors are AAVS serotype capsid vectors.

20. The self-inactivating CRISPR-Cas system of claim 10 , wherein the vector is an AAV vector.

21. The self-inactivating CRISPR-Cas system of claim 20 , wherein the AAV vector is an AAV5 serotype capsid vector.

22. A kit for treating a patient with autosomal dominant Retinitis Pigmentosa in vivo, the kit comprising:

the self-inactivating CRISPR-Cas system of claim 1 ; and

optionally, one or more donor template.

23. A therapeutic for treating a patient with autosomal dominant Retinitis Pigmentosa, the therapeutic comprising the self-inactivating CRISPR-Cas system of claim 1 .

24. The self-inactivating CRISPR-Cas system of claim 1 , wherein the gRNA or the sgRNA is complementary to the genomic target sequence except for in at least one location.

25. A self-inactivating CRISPR-Cas system comprising:

a first segment comprising a nucleotide sequence that encodes Cas9;

a second segment comprising a nucleotide sequence that encodes a gRNA or a sgRNA wherein the gRNA or the sgRNA comprises SEQ ID NO: 5290 or 5291; and

one or more third segments comprising a SIN site, wherein the SIN site is:

a 5′ SIN site located upstream of the nucleotide sequence that encodes Cas9 and upstream of a SV40 NLS within a 5′ UTR; or

a 3′ SIN site located within a naturally occurring or chimeric inserted intron located within the nucleotide sequence that encodes Cas9;

wherein the gRNA or sgRNA is complementary to the SIN site;

wherein the gRNA or sgRNA is complementary to a genomic target sequence.

26. The self-inactivating CRISPR-Cas system of claim 25 , wherein the gRNA or the sgRNA is complementary to the nucleotide sequence of the SIN site except for in at least one location.

27. The self-inactivating CRISPR-Cas system of claim 25 , wherein the gRNA or the sgRNA is complementary to the genomic target sequence except for in at least one location.

28. A method for editing a P23H mutation within a RHO gene, the method comprising: administering the self-inactivating CRISPR-Cas system of claim 1 .

29. A method for treating a patient with a P23H mutation within a RHO gene, the method comprising: administering the self-inactivating CRISPR-Cas system of claim 1 .

30. A method of controlling Cas9 expression in a cell comprising: contacting the cell with the self-inactivating CRISPR-Cas system of claim 1 .