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 .- 20 . (canceled)
21 . A nucleic acid-guided nuclease system comprising:
(a) (i) a nucleic acid-guided nuclease comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, or (ii) a nucleic acid molecule encoding the nucleic acid-guided nuclease of (i); and
(b) (i) an engineered guide nucleic acid capable of complexing with the nucleic acid-guided nuclease, or (ii) a nucleic acid molecule encoding the engineered guide nucleic acid of (i), wherein the engineered guide nucleic acid recognizes a target region in a genome of a cell and a protospacer adjacent motif (PAM) sequence of TTTN.
22 . The nucleic acid-guided nuclease system of claim 21 , wherein the nucleic acid-guided nuclease is encoded by a nucleic acid molecule having at least 80% sequence identity to the nucleotide sequences of SEQ ID NO: 44 or SEQ ID NO: 24.
23 . The nucleic acid-guided nuclease system of claim 21 , wherein the nucleic acid molecule encoding the nucleic acid-guided nuclease is codon optimized for Escherichia Coli.
24 . The nucleic acid-guided nuclease system of claim 21 , wherein the nucleic acid molecule encoding the nucleic acid-guided nuclease is codon optimized for Saccharomyces cerevisiae.
25 . The nucleic acid-guided nuclease system of claim 21 , wherein the nucleic acid molecule encoding the nucleic acid-guided nuclease is codon optimized for mammalian cells.
26 . The nucleic acid-guided nuclease system of claim 21 , wherein the target region is within a coding region of a protein.
27 . The nucleic acid-guided nuclease system of claim 21 , wherein the target region is within a non-coding region of a protein.
28 . The nucleic acid-guided nuclease system of claim 21 , wherein the nucleic acid-guided nuclease comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4.
29 . The nucleic acid-guided nuclease system of claim 21 , wherein the nucleic acid-guided nuclease comprises the amino acid sequence of SEQ ID NO: 4.
30 . 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 21 ; and
(b) allowing the nucleic-acid guided nuclease system to create a genome edit in a target region of the genome of the cell.
31 . The method of claim 30 , wherein the method results in cell death.
32 . The method of claim 30 , wherein the nucleic acid-guided nuclease is encoded by a nucleic acid molecule having at least 80% sequence identity to the nucleotide sequences of SEQ ID NO: 44 or SEQ ID NO: 24.
33 . The method of claim 30 , wherein the nucleic acid-guided nuclease is encoded by a nucleic acid molecule having at least 85% sequence identity to the nucleotide sequences of SEQ ID NO: 44 or SEQ ID NO: 24.
34 . The method of claim 30 , wherein the nucleic acid molecule encoding the nucleic acid-guided nuclease is codon optimized for bacteria.
35 . The method of claim 30 , wherein the nucleic acid molecule encoding the nucleic acid-guided nuclease is codon optimized for mammalian cells.
36 . The method of claim 30 , wherein the nucleic acid-guided nuclease comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4.
37 . The method of claim 30 , wherein the nucleic acid-guided nuclease comprises the amino acid sequence of SEQ ID NO: 4.
38 . The method of claim 30 , wherein the target region is within a bacterial cell.
39 . The method of claim 30 , wherein the target region is within a plant cell.
40 . The method of claim 30 , wherein the target region is within a mammalian cell.