Variants of CRISPR from
Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with altered and improved target specificity and their use in genomic engineering, epigenomic engineering, genome targeting, genome editing, and in vitro diagnostics.
1. An isolated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from Prevotella and Francisella 1 (Cpfl) protein, wherein the protein is from Acidaminococcus sp. BV3L6 (AsCpfl), comprising a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2, with a mutation of N282A in SEQ ID NO:2, wherein the AsCpfl protein is capable of interacting with a guide RNA and target DNA.
2. The isolated protein of claim 1 , wherein the protein further comprises one or more mutations that decrease nuclease activity selected from the group consisting of mutations at D908 and/or E993.
3. The isolated protein of claim 2 , wherein the protein comprises mutations D908A and/or E993A.
4. A fusion protein comprising the isolated protein of claim 1 , fused to a heterologous functional domain, with an optional intervening linker, wherein the AsCpfl protein optionally comprises mutations D908A and/or E993A, and the AsCpfl protein is capable of interacting with a guide RNA and target DNA.
5. The fusion protein of claim 4 , wherein the heterologous functional domain is a transcriptional activation domain.
6. The fusion protein of claim 5 , wherein the transcriptional activation domain is from VP64 or NF- k B p65.
7. The fusion protein of claim 4 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.
8. The fusion protein of claim 7 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).
9. The fusion protein of claim 7 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).
10. The fusion protein of claim 4 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.
11. The fusion protein of claim 10 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.
12. The fusion protein of claim 11 , wherein the TET protein is TET1.
13. The fusion protein of claim 4 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.
14. The fusion protein of claim 13 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.
15. The fusion protein of claim 4 , wherein the heterologous functional domain is a biological tether.
16. The fusion protein of claim 15 , wherein the biological tether is MS2, Csy4 or lambda N protein.
17. The fusion protein of claim 4 , wherein the heterologous functional domain is FokI.
18. The isolated protein of claim 1 , wherein the protein comprises SEQ ID NO:2, except having the mutation of N282A in SEQ ID NO:2 and at least 10 additional amino acid substitutions.
19. The isolated protein of claim 18 , wherein the protein comprises SEQ ID NO:2, except having the mutation of N282A in SEQ ID NO:2.
20. An isolated nucleic acid encoding an isolated Cpfl protein, wherein the protein is from Acidaminococcus sp. BV3L6 (AsCpfl), comprising a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2, with a mutation at position N282A of SEQ ID NO:2, wherein the AsCpfl protein is capable of interacting with a guide RNA and target DNA.
21. A vector comprising the isolated nucleic acid of claim 20 .
22. An isolated host cell comprising the isolated nucleic acid of claim 20 .
23. The isolated nucleic acid of claim 20 , wherein the nucleic acid encodes the protein comprising SEQ ID NO:2, except having the mutation of N282A in SEQ ID NO:2 and at least 10 additional amino acid substitutions.
24. The isolated nucleic acid of claim 23 , wherein the nucleic acid encodes the protein comprising SEQ ID NO:2, except having the mutation of N282A in SEQ ID NO:2.