Novel guide rna/cas endonuclease systems

Abstract

Compositions and methods are provided for novel guide RNA/Cas endonuclease systems. Type II Cas9 endonuclease systems originating from Brevibacillus laterosporus, Lactobacillus reuteri Mlc3, Lactobacillus rossiae DSM 15814, Pediococcus pentosaceus SL4, Lactobacillus nodensis JCM 14932, Sulfurospirillum sp. SCADC, Bifidobacterium thermophilum DSM 20210, Loktanella vestfoldensis, Sphingomonas sanxanigenens NX02, Epilithonimonas tenax DSM 16811, Sporocytophaga myxococcoides are described herein. The present disclosure also describes methods for genome modification of a target sequence in the genome of a cell, for gene editing, and for inserting a polynucleotide of interest into the genome of a cell.

1 .- 17 . (canceled) 
     
     
         18 . A guide RNA/Cas9 endonuclease complex comprising at least one of the following:
 a. a guide RNA, wherein the guide RNA is a duplex molecule comprising a chimeric non-naturally occurring crRNA and a tracrRNA, wherein said chimeric non-naturally occurring crRNA comprises a variable targeting domain capable of hybridizing to said target sequence, wherein said tracrRNA originated from  Brevibacillus laterosporus;      b. a guide RNA, wherein the guide RNA is a duplex molecule comprising a chimeric non-naturally occurring crRNA and a tracrRNA, wherein said chimeric non-naturally occurring crRNA comprises at least a fragment of a crRNA originating from  Brevibacillus laterosporus ; wherein said chimeric non-naturally occurring crRNA comprises a variable targeting domain capable of hybridizing to said target sequence; or   c. a Cas9 endonuclease originating from  Brevibacillus laterosporus;      wherein said guide RNA/Cas9 endonuclease complex is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a target sequence.   
     
     
         19 . The guide RNA/Cas9 endonuclease complex of  claim 18 , wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47, 68, 143, 145, 147, 195, 197, and 199. 
     
     
         20 . The guide RNA/Cas9 endonuclease complex of  claim 18 , wherein the Cas9 endonuclease is SEQ ID NO: 140, or a functional fragment thereof, or encoded by SEQ ID NO: 36. 
     
     
         21 . A method for modifying a target site in the genome of a cell, the method comprising providing to said cell at least one guide RNA and one Cas9 endonuclease, wherein said guide RNA and Cas9 endonuclease can form a complex that is capable of recognizing, binding to, and optionally nicking or cleaving all or part of said target site; wherein the Cas9 endonuclease originated from  Brevibacillus laterosporus.    
     
     
         22 . The method of  claim 21 , further comprising providing to said cell a polynucleotide modification template. 
     
     
         23 . The method of  claim 21 , further comprising providing to said cell at least one donor DNA, wherein said donor DNA comprises a polynucleotide of interest. 
     
     
         24 . The method of  claim 21 , wherein the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 47, 68, 143, 145, 147, 195, 197, and 199. 
     
     
         25 . The method of  claim 21 , wherein the Cas9 endonuclease is SEQ ID NO: 140, or a functional fragment thereof, or encoded by SEQ ID NO: 36. 
     
     
         26 . The method of  claim 21 , further comprising identifying at least one cell that has a modification at said target site, wherein the modification at said target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). 
     
     
         27 . The method of  claim 23 , further comprising identifying at least one cell that said polynucleotide of interest integrated in or near said target site. 
     
     
         28 . The method of any one of  claims 21 - 27 , wherein the cell is selected from the group consisting of a human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cell. 
     
     
         29 . A kit for binding, cleaving, or nicking a target sequence in eukaryotic cells or organisms, comprising a Cas9 endonuclease protein obtained from  Brevibacillus laterosporus ; wherein the Cas9 endonuclease is capable of forming a complex with a guide RNA, wherein the complex can recognize the target sequence. 
     
     
         30 . The kit of  claim 29 , wherein the Cas9 endonuclease is SEQ ID NO: 140, or a functional fragment thereof, or encoded by SEQ ID NO: 36. 
     
     
         31 . The kit of  claim 29 , wherein the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 47, 68, 143, 145, 147, 195, 197, and 199. 
     
     
         32 . The kit of  claim 29 , further comprising a molecule selected from the group consisting of an inhibitor of NHEJ, an activator of HDR or MMEJ repair pathways, an exogenous sequence, a homologous recombination DNA, a polynucleotide modification template, a donor DNA, and any combination thereof.