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) (i) a nucleic acid-guided nuclease comprising at least 90% amino acid sequence identity to SEO ID NO: 7, or (ii) a nucleic acid molecule encoding the nucleic acid-guided nuclease of (i); and
(b) (i) an engineered guide nucleic acid comprising a region for complexing with the nucleic acid-guided nuclease and a region for hybridizing with a target sequence, thereby directing the nucleic acid-guided nuclease to the target sequence, or (ii) a nucleic acid molecule encoding the engineered guide nucleic acid of (i).
2. The system of claim 1 , wherein the region for binding complexing to the nucleic acid-guided nuclease is a pseudoknot region.
3. The system of claim 2 , wherein the region for complexing with the nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NO: 88, SEQ ID NO: 93, and SEQ ID NO: 94.
4. The system of claim 1 , wherein the target sequence is within a eukaryotic cell.
5. The system of claim 1 , wherein the target sequence is within a bacterial cell.
6. The system of claim 1 , wherein the target sequence is within a plant cell.
7. The system of claim 1 , wherein the target sequence is within a mammalian cell.
8. The system of claim 1 , wherein the system has an editing efficiency of from 97% to 100% efficiency as measured by dividing the number of edited cells over a total number of recovered cells.
9. The system of claim 1 , further comprising an editing sequence having a change in sequence relative to the sequence of a target region.
10. The system of claim 1 , wherein the nucleic acid-guided nuclease comprises at least 95% amino acid sequence identity to SEQ ID NO: 7.
11. The system of claim 1 , wherein the nucleic acid-guided nuclease directs cleavage of one nucleic acid strand at the target sequence.
12. The system of claim 1 , wherein the nucleic acid-guided nuclease directs cleavage of two nucleic acid strands at the target sequence.
13. The system of claim 1 , wherein the engineered guide nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 90.
14. The system of claim 1 , wherein the engineered guide nucleic acid comprises a pseudoknot region comprising nucleotide residues 18-26 of SEQ ID NO: 93.
15. The system of claim 1 , wherein the engineered guide nucleic acid comprises a pseudoknot region comprising a sequence selected from the group consisting of nucleotide residues 21-36 of SEQ ID NO: 88, nucleotide residues 19-34 of SEQ ID NO: 93, and nucleotide residues 19-34 of SEQ ID NO: 94.
16. The system of claim 9 , wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid.
17. The system of claim 16 , wherein the editing sequence further comprises a mutation in a protospacer adjacent motif (PAM) site.