Materials and methods for controlling gene editing

1. A CRISPR/Cas system comprising:

a nuclease segment comprising a codon optimized nucleotide sequence that encodes a Cas9 nuclease or variant thereof, wherein the Cas9 nuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60;

a guide RNA (gRNA) segment comprising a nucleotide sequence that encodes a gRNA or sgRNA;

a promoter segment comprising a nucleotide sequence that encodes a promoter comprising one or more tetracycline operator sequence, wherein the gRNA segment is operably linked to the promoter segment; and/or

a short-hairpin RNA (shRNA) segment comprising a nucleotide sequence that encodes a shRNA that comprises sequence that is complementary to a transcript from the nuclease segment;

wherein the Cas9 nuclease comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60 introduces a double stranded break at a site in a nucleic acid complementary to the gRNA segment.

2. The CRISPR/Cas system of claim 1 , wherein the promoter is selected from a group consisting of: H1 promoter, U6 promoter, 7SK promoter, and portions of any thereof.

3. The CRISPR/Cas system of claim 1 , wherein the one or more tetracycline operator sequence comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 4, or comprises SEQ ID NO: 4.

4. The CRISPR/Cas system of claim 1 , further comprising:

a repressor segment comprising a nucleotide sequence that encodes a tetracycline repressor protein.

5. The CRISPR/Cas system of claim 4 , wherein the tetracycline repressor comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 62, or comprises SEQ ID NO: 62.

6. The CRISPR/Cas system of claim 1 , wherein the one or more tetracycline operator sequence is capable of being bound by the tetracycline repressor protein.

7. The CRISPR/Cas system of claim 1 , further comprising:

one or more self-inactivating segments comprising a SIN site;

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

wherein the gRNA or sgRNA is substantially complementary to a genomic target sequence within a cell of a patient.

8. The CRISPR/Cas system of claim 7 , wherein the one or more self-inactivating segments are located in at least one of:

(i) at the 5′ end of the codon optimized nucleotide sequence that encodes the Cas9 nuclease or variant thereof;

(ii) at the 3′ end of the codon optimized nucleotide sequence that encodes the Cas9 nuclease or variant thereof; and

(iii) in an intron within the codon optimized nucleotide sequence that encodes the Cas9 nuclease or variant thereof.

9. The CRISPR/Cas system of claim 7 , wherein one of the one or more self-inactivating segments are located upstream of the codon optimized nucleotide sequence that encodes the Cas9 nuclease or variant thereof and downstream of a nuclear localization signal (NLS).

10. The CRISPR/Cas system of claim 7 , wherein the SIN site comprises a protospacer adjacent motif (PAM) sequence.

11. The CRISPR/Cas system of claim 10 , wherein the PAM sequence in the SIN site is NNGG.

12. The CRISPR/Cas system of claim 1 , wherein the gRNA or sgRNA is fully complementary to the nucleotide sequence of the SIN site except for at one base pair or except for at two base pairs.

13. The CRISPR/Cas system of claim 1 , further comprising a nucleic acid sequence encoding a promoter that is operably linked to the codon optimized nucleotide sequence that encodes the Cas9 nuclease or variant thereof.

14. The CRISPR/Cas system of claim 13 , wherein the promoter is a spatially-restricted promoter, bidirectional promoter, or an inducible promoter.

15. The CRISPR/Cas system of claim 14 , wherein the spatially-restricted promoter is selected from a group consisting of: 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.

16. The CRISPR/Cas system of claim 1 , wherein the gRNA or sgRNA comprises a spacer sequence comprising 17 to 24 nucleotides.

17. The CRISPR/Cas system of claim 1 , wherein

(i) the nuclease segment is provided in a first vector, and the gRNA segment and the promoter segment are provided together in a second vector or

(ii) the nuclease segment and the gRNA segment are provided together in a first vector and the shRNA segment is provided in a second vector.

18. The CRISPR/Cas system of claim 4 , wherein

(i) the nuclease segment, the gRNA segment, and the promoter segment are provided together in a first vector and the repressor segment is provided in a second vector,

(ii) the nuclease segment is provided in a first vector, the gRNA segment and the promoter segment are provided together in a second vector, and the repressor segment and/or the shRNA segment is provided in a third vector; or

the nuclease segment, the gRNA segment, and the promoter segment are provided together in a first vector and the repressor segment and/or the shRNA segment is provided in a second vector.

19. The CRISPR/Cas system of claim 7 , wherein

(i) the nuclease segment, the gRNA segment, the promoter segment, and the one or more self-inactivating segments are provided together in a first vector and the repressor segment is provided in a second vector,

(ii) the nuclease segment, the gRNA segment, the promoter segment, the one or more self-inactivating segments, and the repressor segment are provided in a vector;

(iv) the nuclease segment, the gRNA segment, and the one or more self-inactivating segments are provided together in a first vector and the shRNA segment is provided in a second vector; or

(v) the nuclease segment, the gRNA segment, the one or more self-inactivating segments, and the shRNA segment are provided in a vector.

20. The CRISPR/Cas system of claim 17 , wherein the first vector and the second vector are AAV vectors or plasmids.

21. The CRISPR/Cas system of claim 20 , wherein the AAV vectors are AAV2 serotype vectors, AAV5 serotype vectors, or AAV6 serotype vectors.

22. A pharmaceutical composition comprising the CRISPR/Cas system of claim 1 .

23. A packaging cell comprising the CRISPR/Cas system of claim 1 .

24. A recombinant AAV vector comprising:

a nuclease segment comprising a codon optimized nucleotide sequence that encodes a Cas9 nuclease or variant thereof; wherein the Cas9 nuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60, wherein the Cas9 nuclease comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60 introduces a double stranded break at a site in a nucleic acid complementary to the gRNA segment;

a gRNA segment comprising a nucleotide sequence that encodes a gRNA or sgRNA; and

a promoter segment comprising a nucleotide sequence that encodes a first promoter comprising one or more tetracycline operator sequence, wherein the gRNA segment is operably linked to the promoter segment.

25. The recombinant AAV vector of claim 24 , wherein the recombinant AAV vector comprises a nucleic acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 66-69.

26. A pharmaceutical composition comprising the recombinant AAV vector of claim 24 .

27. An isolated genetically modified cell comprising the recombinant AAV vector of claim 24 .

28. The CRISPR/Cas system of claim 1 , wherein the shRNA comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs 55¬59, or the shRNA comprises any one of SEQ ID NOs: 9-11.