US2020231987A1PendingUtilityA1

Nucleic acid-guided nucleases

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Assignee: INSCRIPTA INCPriority: Jun 23, 2017Filed: Mar 16, 2020Published: Jul 23, 2020
Est. expiryJun 23, 2037(~10.9 yrs left)· nominal 20-yr term from priority
Inventors:Andrew GarstRyan T. GillTanya Elizabeth Warnecke Lipscomb
C12N 15/8509C12N 2310/20C12N 15/70C12N 15/81C12N 15/111C12N 9/22C12N 2800/22C12N 15/11C12N 15/905C12N 15/902C12N 15/907C12N 15/85
74
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Claims

Abstract

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.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method of modifying a target region in the genome of a cell, the method comprising:
 (a) contacting a cell with:   a non-naturally occurring nucleic acid-guided nuclease encoded by a nucleic acid having at least 90% identity to SEQ ID No. 22, SEQ ID No. 42, SEQ ID No. 128 or SEQ ID No. 148;   an engineered guide nucleic acid capable of complexing with the nucleic acid-guided nuclease; and   an editing sequence encoding a nucleic acid complementary to the target region having a change in sequence relative to the target region and a mutation in a protospacer adjacent motif (PAM) sequence; and   (b) allowing the nuclease, guide nucleic acid and editing sequence to create a genome edit in a target region of the genome of the cell.   
     
     
         2 . The method of  claim 1 , wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein the engineered guide nucleic acid comprises SEQ ID No. 88, SEQ ID No. 93 or SEQ ID No. 94. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid-guided has less than 40% protein identity to SEQ ID No. 12 and SEQ ID No. 108. 
     
     
         5 . The method of  claim 1 , wherein the nucleic acid-guided nuclease is codon optimized for the cell to be edited. 
     
     
         6 . The method of  claim 5 , wherein the nucleic acid-guided nuclease is codon optimized for  E. coli.    
     
     
         7 . The method of  claim 5 , wherein the nucleic acid-guided nuclease is codon optimized for  S. cerevisiae.    
     
     
         8 . The method of  claim 5 , wherein the nucleic acid-guided nuclease is codon optimized for mammalian cells. 
     
     
         9 . The method of  claim 1 , wherein the non-naturally occurring nucleic acid-guided nuclease is encoded by a nucleic acid having at least 95% identity to SEQ ID No. 22, SEQ ID No. 42, SEQ ID No. 128 or SEQ ID No. 148. 
     
     
         10 . The method of  claim 1 , wherein the non-naturally occurring nucleic acid-guided nuclease is encoded by a nucleic acid having greater than 95% identity to SEQ ID No. 22, SEQ ID No. 42, SEQ ID No. 128 or SEQ ID No. 148. 
     
     
         11 . A nucleic acid-guided nuclease system comprising:
 (a) a non-naturally occurring nuclease encoded by a nucleic acid having at least 90% identity to SEQ ID No. 22, SEQ ID No. 42, SEQ ID No. 128 or SEQ ID No. 148;   (b) an engineered guide nucleic acid capable of complexing with the nucleic acid-guided nuclease, and   (c) an editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell and a mutation in a protospacer adjacent motif (PAM) sequence; wherein the system results in a genome edit in the target region of the genome of the cells facilitated by the nuclease, the engineered guide nucleic acid and the editing sequence.   
     
     
         12 . The system of  claim 11 , wherein the engineered guide nucleic acid comprises SEQ ID No. 88, SEQ ID No. 93 or SEQ ID No. 94. 
     
     
         13 . The system of  claim 11 , wherein the nucleic acid-guided has less than 40% protein identity to SEQ ID No. 12 and SEQ ID No. 108. 
     
     
         14 . The system of  claim 11 , wherein the nucleic acid-guided nuclease is codon optimized for the cell to be edited. 
     
     
         15 . The system of  claim 14 , wherein the nucleic acid-guided nuclease is codon optimized for  E. coli.    
     
     
         16 . The system of  claim 14 , wherein the nucleic acid-guided nuclease is codon optimized for  S. cerevisiae.    
     
     
         17 . The system of  claim 14 , wherein the nucleic acid-guided nuclease is codon optimized for mammalian cells. 
     
     
         18 . The system of  claim 11 , wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid. 
     
     
         19 . The system of  claim 11 , wherein the non-naturally occurring nucleic acid-guided nuclease is encoded by a nucleic acid having at least 95% identity to SEQ ID No. 22, SEQ ID No. 42, SEQ ID No. 128 or SEQ ID No. 148. 
     
     
         20 . The system of  claim 11 , wherein the non-naturally occurring nucleic acid-guided nuclease is encoded by a nucleic acid having at least 95% identity to SEQ ID No. 22, SEQ ID No. 42, SEQ ID No. 128 or SEQ ID No. 148

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