Constructs, compositions and methods thereof having improved genome editing efficiency and specificity

Abstract

Embodiments disclosed herein include novel nucleic acid-guided nucleases, novel guide nucleic acids, and novel targetable nuclease systems, and methods of use. In some embodiments, engineered non-naturally occurring nucleic acid-guided nucleases, can be used with known guide nucleic acids in a targetable nuclease system. In certain embodiments, targetable nuclease systems can be used to edit targeted genomes of humans and other species. In some embodiments, methods include, but are not limited to, recursive genetic engineering and trackable genetic engineering methods.

What is claimed is: 
     
         1 . A nucleic acid-guided nuclease system comprising:
 (a) an engineered nucleic acid-guided nuclease, wherein the engineered nucleic acid-guided nuclease comprises a polypeptide sequence comprising at least 85% homology to the polypeptide represented by SEQ ID NO: 94 (ABW8), 29 (ABW3), 81 (ABW7), 107 (ABW9), 3 (ABW1), 16 (ABW2), 42 (ABW4), 55 (ABW5), and 68 (ABW6); or at least 85% homology to the polynucleotide encoding a polypeptide comprising a polynucleotide represented by SEQ ID NO: 95-104 (ABW8 variants 1-10), 30-39 (ABW3 variants 1-10), 82-91 (ABW7 variants 1-10), 108-117 (ABW9 variants 1-10), 4-13 (ABW1 variants 1-10), 17-26 (ABW2 variants 1-10), 43-52 (ABW4 variants 1-10), 56-65 (ABW5 variants 1-10), and 69-78 (ABW6 variants 1-10); and   (b) a guide polynucleotide for complexing with the nucleic acid-guided nuclease.   
     
     
         2 . The system according to  claim 1 , wherein the target region is within a eukaryotic cell. 
     
     
         3 . The system according to  claim 1 , wherein the target region is within a bacterial cell. 
     
     
         4 . The system according to  claim 1 , wherein the target region is within a plant cell. 
     
     
         5 . The system according to  claim 1 , wherein the target region is within a mammalian cell. 
     
     
         6 . The system according to any one of  claims 1 - 5 , wherein the gRNA comprises STAR gRNA. 
     
     
         7 . The system according to any one of  claims 1 - 5 , wherein the gRNA comprises split gRNA. 
     
     
         8 . A nucleic acid-guided nuclease system comprising:
 (a) a nucleic acid-guided nuclease; and   (b) an engineered guide polynucleotide (gRNA) for complexing with the nucleic acid-guided nuclease, wherein the engineered guide polynucleotide comprises a polynucleotide represented by SEQ ID NO: 125, 120, 124, 126, 118, 119, 121, 122, 123, 127, or 128.   
     
     
         9 . The system according to  claim 8 , wherein the target region is within a eukaryotic cell. 
     
     
         10 . The system according to  claim 8 , wherein the target region is within a bacterial cell. 
     
     
         11 . The system according to  claim 8 , wherein the target region is within a plant cell. 
     
     
         12 . The system according to  claim 8 , wherein the target region is within a mammalian cell. 
     
     
         13 . The system according to  claim 8 , wherein the target region is within a human cell. 
     
     
         14 . The system according to any one of  claims 8 - 13 , wherein the gRNA comprises STAR gRNA. 
     
     
         15 . The system according to any one of  claims 8 - 13 , wherein the gRNA comprises split gRNA. 
     
     
         16 . A nucleic acid-guided nuclease system comprising:
 (a) an engineered nucleic acid-guided nuclease, wherein the engineered nucleic acid-guided nuclease comprises a polypeptide sequence comprising at least 85% homology to the polypeptide represented by SEQ ID NO: 94 (ABW8), 29 (ABW3), 81 (ABW7), 107 (ABW9), 3 (ABW1), 16 (ABW2), 42 (ABW4), 55 (ABW5), and 68 (ABW6), and   (b) an engineered guide polynucleotide (gRNA) for complexing with the nucleic acid-guided nuclease, wherein the engineered guide polynucleotide comprises a polynucleotide represented by SEQ ID NO: 125, 120, 124, 126, 118, 119, 121, 122, 123, 127 or 128.   
     
     
         17 . The system according to  claim 16 , wherein the target region is within a eukaryotic cell. 
     
     
         18 . The system according to  claim 16 , wherein the target region is within a bacterial cell. 
     
     
         19 . The system according to  claim 16 , wherein the target region is within a plant cell. 
     
     
         20 . The system according to  claim 16 , wherein the target region is within a mammalian cell. 
     
     
         21 . A method of modifying a target region, the method comprising:
 (a) contacting a sample having a targeted genomic region with:
 (i) an engineered nucleic acid-guided nuclease according to  claim 1 ; and 
 (ii) a guide nucleic acid according to any one of  claim 1  or  8 ; and 
   (b) allowing the nuclease and the guide nucleic acid to modify the target region.   
     
     
         22 . The method according to  claim 21 , wherein the sample is further contacted with (iii) an editing sequence having a change in sequence relative to the sequence of the targeted genomic region. 
     
     
         23 . The method according to  claim 21 , wherein modifying the targeted genomic region comprises editing the targeted genomic region. 
     
     
         24 . The method according to  claim 21 , wherein the guide nucleic acid (ii) and the editing sequence (iii) are provided as a single nucleic acid. 
     
     
         25 . The method according to  claim 21 , wherein the editing sequence further comprises a mutation in a protospacer adjacent motif (PAM) site. 
     
     
         26 . The method according to  claim 21 , wherein the target region is within a eukaryotic cell. 
     
     
         27 . The method according to  claim 21 , wherein the target region is within a bacterial cell. 
     
     
         28 . The method according to  claim 21 , wherein the target region is within a plant cell. 
     
     
         29 . The method according to  claim 21 , wherein the target region is within a mammalian cell. 
     
     
         30 . A kit comprising:
 (a) an engineered nucleic acid-guided nuclease, wherein the engineered nucleic acid-guided nuclease comprises a polypeptide sequence comprising at least 85% homology to the polypeptide represented by SEQ ID NO: 95-104 (ABW8 variants 1-10), 30-39 (ABW3 variants 1-10), 82-91 (ABW7 variants 1-10), 108-117 (ABW9 variants 1-10), 4-13 (ABW1 variants 1-10), 17-26 (ABW2 variants 1-10), 43-52 (ABW4 variants 1-10), 56-65 (ABW5 variants 1-10), and 69-78 (ABW6 variants 1-10) and, optionally,   (b) an engineered guide polynucleotide for complexing with the nucleic acid-guided nuclease; optionally, wherein the engineered guide polynucleotide comprises a polynucleotide represented by SEQ ID NO: 125, 120, 124, 126, 118, 119, 121, 122, 123, 127 or 128; and   (c) a container.   
     
     
         31 . The kit according to  claim 30 , wherein the kit is of use to edit a prokaryote genome, a plant genome, a eukaryotic genome or yeast genome. 
     
     
         32 . The kit according to  claim 30 , wherein the kit is of use to edit a eukaryotic genome of a human or other mammal.