Dual strand nucleic acid-guided nickase editing

Abstract

The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion enzyme editing of nucleic acids in live mammalian cells.

We claim: 
     
         1 . A method for performing nucleic acid-guided nickase/reverse transcriptase fusion editing in a target locus in a genome of a live cell editing utilizing two CREATE Fusion (“CF”) editing cassettes comprising:
 providing a cell with the target locus; 
 providing a nucleic acid-guided nuclease/reverse transcriptase fusion enzyme; 
 providing first and second CF editing cassettes, wherein the first and second CF editing cassettes have regions of complementarity to opposite strands of the target locus, the first CF editing cassette comprising from 5′ to 3′:
 a first CFgRNA comprising a 5′ to 3′spacer region and scaffold region; and 
 a first repair template comprising an optional post-edit homology (PEH) region, an edit, a nick-to-edit region, and a primer binding site (PBS) region; and 
 
 the second CF editing cassette comprising from 5′ to 3′:
 a second CFgRNA comprising a 5′ to 3′spacer region and scaffold region; and 
 a second repair template comprising an optional post-edit homology (PEH) region, an edit, a nick-to-edit region, and a primer binding site (PBS) region; 
 
 providing conditions to allow the nucleic acid-guided nuclease/reverse transcriptase fusion enzyme and CF editing cassettes to bind to the opposite strands of the target locus; and 
 allowing the nucleic acid-guided nuclease/reverse transcriptase fusion enzyme and CF editing cassettes to edit the target locus. 
 
     
     
         2 . The method of  claim 1 , wherein the CF editing cassettes further comprise an edit to immunize the target locus to prevent re-nicking. 
     
     
         3 . The method of  claim 1 , wherein the nick-to-edit region of at least one of the first or second CF editing cassette is from 2-250 nucleotides in length. 
     
     
         4 . The method of  claim 3 , wherein the nick-to-edit region of at least one of the first or second CF editing cassette is from 5-150 nucleotides in length. 
     
     
         5 . The method of  claim 1 , wherein the nick-to-edit region of at least one of the first or second CF editing cassette is from 0-150 nucleotides in length. 
     
     
         6 . The method of  claim 1 , wherein the region of complementarity between the first and second CF editing cassettes is from 4-120 nucleotides in length. 
     
     
         7 . The method of  claim 6 , wherein the region of complementarity between the first and second CF editing cassettes is from 5-80 nucleotides in length. 
     
     
         8 . The method of  claim 7 , wherein the region of complementarity between the first and second CF editing cassettes is from 6-60 nucleotides in length. 
     
     
         9 . The method of  claim 1 , wherein the edit region of the first and second CF editing cassettes is from 1-750 nucleotides in length. 
     
     
         10 . The method of  claim 9 , wherein the edit region of the first and second CF editing cassettes is from 1-500 nucleotides in length. 
     
     
         11 . The method of  claim 10 , wherein the edit region of the first and second CF editing cassettes is from 1-150 nucleotides in length. 
     
     
         12 . The method of  claim 1 , wherein the post-edit homology region of at least one of the first or second CF editing cassettes is from 2-50 nucleotides in length. 
     
     
         13 . The method of  claim 12 , wherein the post-edit homology region of at least one of the first or second CF editing cassettes is from 4-40 nucleotides in length. 
     
     
         14 . The method of  claim 13 , wherein the post-edit homology region of at least one of the first or second CF editing cassettes is from 5-25 nucleotides in length. 
     
     
         15 . The method of  claim 1 , wherein the edit is a single base swap in the target locus. 
     
     
         16 . The method of  claim 1 , wherein the edit is an insertion in the target locus. 
     
     
         17 . The method of  claim 1 , wherein the edit is a deletion in the target locus. 
     
     
         18 . The method of  claim 1 , wherein the edit is in a coding region in the target locus. 
     
     
         19 . The method of  claim 1 , wherein the edit is in a noncoding region in the target locus. 
     
     
         20 . The method of  claim 1 , wherein the nuclease portion of the nickase/reverse transcriptase fusion is a MAD2007 nickase.