Engineered enzyme
The present disclosure provides a novel catalytically inactive MAD7 nuclease (dMAD7) that retains the ability to bind DNA in a sequence-specific manner. The MAD7 nuclease from which the dMAD7 has been derived was isolated from Eubacterium rectale.
1. A catalytically inactive CRISPR nuclease comprising a variant amino acid sequence of SEQ ID NO: 1, and wherein the variant amino acid sequence comprises an alanine residue at amino acid position 877 as compared to SEQ ID NO: 1.
2. The nuclease of claim 1 , wherein the nuclease binds a bacteria genome in a sequence-specific manner.
3. The nuclease of claim 1 , wherein the nuclease binds a yeast genome in a sequence-specific manner.
4. The nuclease of claim 1 , wherein the nuclease binds a mammalian genome in a sequence specific manner.
5. A vector comprising a nucleic acid sequence encoding the nuclease of claim 1 .
6. The vector of claim 5 , wherein the vector further comprises a nucleic acid sequence encoding a guide RNA.
7. The vector of claim 5 , wherein the vector further comprises a nucleic acid sequence encoding a donor nucleic acid.
8. The vector of claim 5 , wherein the nucleic acid sequence encoding the nuclease further comprises a nucleic acid sequence encoding one or more nuclear localization sequences.
9. The vector of claim 5 , wherein the nucleic acid sequence encoding the nuclease is operably linked to a promoter.
10. The vector of claim 9 , wherein the promoter is an inducible promoter.
11. The vector of claim 9 , wherein the promoter is a constitutive promoter.
12. The vector of claim 6 , wherein the nucleic acid sequence encoding a guide RNA is operably linked to a promoter.
13. The vector of claim 12 , wherein the promoter is an inducible promoter.
14. The vector of claim 12 , wherein the promoter is a constitutive promoter.
15. The vector of claim 9 , wherein the nucleic acid sequence encoding the nuclease is codon-optimized for expression in a eukaryotic cell.
16. The vector of claim 15 , wherein the eukaryotic cell is a fungal cell.
17. The vector of claim 16 , wherein the fungal cell is a yeast cell.
18. The vector of claim 15 , wherein the eukaryotic cell is a human cell.
19. The vector of claim 15 , wherein the eukaryotic cell is a plant cell.
20. The vector of claim 9 , wherein the nucleic acid sequence encoding the nuclease is codon-optimized for expression in a prokaryotic cell.