Nucleic acid-guided nucleases
Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.
1. A nucleic acid-guided nuclease system comprising:
(a) a nucleic acid having at least 95% identity to SEQ ID NO: 24 or SEQ ID NO: 44, wherein the nucleic acid encodes a nucleic acid-guided nuclease comprising the amino acid sequence of SEQ ID No. 4;
(b) an engineered guide nucleic acid sequence capable of complexing with the nucleic acid-guided nuclease, wherein the engineered guide nucleic acid sequence is configured to hybridize to a TTTN sequence of a target region in a genome of a cell;
(c) an editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell;
wherein the system results in a genome edit in the target region in the genome of the cell facilitated by the nuclease, the engineered guide nucleic acid, and the editing sequence.
2. The system of claim 1 , wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid.
3. The system of claim 1 , wherein the nucleic acid encoding the nucleic acid-guided nuclease is codon optimized for E. coli.
4. The system of claim 1 , wherein the nucleic acid encoding the nucleic acid-guided nuclease is codon optimized for S. cerevisiae.
5. The system of claim 1 , wherein the nucleic acid encoding the nucleic acid-guided nuclease is codon optimized for mammalian cells.
6. The system of claim 1 , wherein the target region is within a coding region of a protein.
7. The system of claim 1 , wherein the target region is within a non-coding region of a protein.
8. The system of claim 7 , wherein the target region is within a regulatory region of a protein.
9. The system of claim 1 , wherein the editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell further comprises a mutation in a protospacer adjacent motif (PAM) sequence.
10. A method of modifying a target region in the genome of a cell, the method comprising:
(a) contacting a cell with:
the nucleic acid-guided nuclease system of claim 1 ; and
(b) allowing the nucleic-acid guided nuclease system to create a genome edit in a target region of the genome of the cell.
11. The method of claim 10 , wherein the engineered guide nucleic acid sequence and the editing sequence are provided as a single nucleic acid.
12. The method of claim 1 , wherein the nucleic acid having at least 95% identity to SEQ ID No. 44 or SEQ ID No. 24 is codon optimized for the cell to be edited.
13. The method of claim 12 , wherein the nucleic acid-guided nuclease is codon optimized for E. coli.
14. The method of claim 12 , wherein the nucleic acid-guided nuclease is codon optimized for S. cerevisiae.
15. The method of claim 12 , wherein the nucleic acid-guided nuclease is codon optimized for mammalian cells.
16. The method of claim 10 , wherein the editing sequence encoding a nucleic acid complementary to the target region having a change in sequence relative to the target region further comprises a mutation in a protospacer adjacent motif (PAM) site.
17. The method of claim 10 , wherein the target region is within a eukaryotic cell.
18. The method of claim 10 , wherein the target region is within a bacterial cell.
19. The method of claim 10 , wherein the target region is within a plant cell.
20. The method of claim 10 , wherein the target region is within a mammalian cell.