Engineered CRISPR-Cas9 nucleases with altered PAM specificity
Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.
1. An isolated nucleic acid encoding a variant Streptococcus pyogenes (SpCas9) protein comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, with an amino acid mutation at D1135 and optionally at one or more of the following positions: G1104, 51109, L1111, 51136, G1218, N1317, R1335, or T1337.
2. A vector comprising the isolated nucleic acid of claim 1 .
3. The vector of claim 2 , wherein the isolated nucleic acid is operably linked to one or more regulatory domains for expressing the variant SpCas9 protein.
4. A host cell comprising the nucleic acid of claim 1 .
5. The isolated nucleic acid of claim 1 , wherein the variant SpCas9 protein is fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.
6. The isolated nucleic acid of claim 5 , wherein the heterologous functional domain is a transcriptional activation domain.
7. The isolated nucleic acid of claim 6 , wherein the transcriptional activation domain is from VP64 or NF-κB p65.
8. The isolated nucleic acid of claim 5 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.
9. The isolated nucleic acid of claim 8 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).
10. The isolated nucleic acid of claim 8 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).
11. The isolated nucleic acid of claim 5 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.
12. The isolated nucleic acid of claim 11 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) protein.
13. The isolated nucleic acid of claim 12 , wherein the TET protein is TET1.
14. The isolated nucleic acid of claim 5 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.
15. The isolated nucleic acid of claim 14 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methyltransferase (HMT), or a histone demethylase.
16. The isolated nucleic acid of claim 5 , wherein the heterologous functional domain is a biological tether.
17. The isolated nucleic acid of claim 16 , wherein the biological tether is MS2, Csy4, or lambda N protein.
18. The isolated nucleic acid of claim 5 , wherein the heterologous functional domain is FokI.
19. A composition comprising:
a nucleic acid encoding a variant SpCas9 protein comprising an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, with an amino acid mutation at D1135 and optionally at one or more of the following positions: G1104, 51109, L1111, 51136, G1218, N1317, R1335, T1337; and
a nucleic acid encoding a guide RNA that directs the SpCas9 protein to a target genomic sequence.
20. The composition of claim 19 , wherein the variant SpCas9 protein is fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.
21. The composition of claim 20 , wherein the heterologous functional domain is a transcriptional activation domain.
22. The composition of claim 21 , wherein the transcriptional activation domain is from VP64 or NF-κB p65.
23. The composition of claim 20 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.
24. The composition of claim 23 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).
25. The composition of claim 23 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).
26. The composition of claim 20 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.
27. The composition of claim 26 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) protein.
28. The composition of claim 27 , wherein the TET protein is TET1.
29. The composition of claim 20 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.
30. The composition of claim 29 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methyltransferase (HMT), or a histone demethylase.
31. The composition of claim 20 , wherein the heterologous functional domain is a biological tether.
32. The composition of claim 31 , wherein the biological tether is MS2, Csy4, or lambda N protein.
33. The composition of claim 20 , wherein the heterologous functional domain is FokI.