Rapid characterization of cas endonuclease systems, pam sequences and guide rna elements

Abstract

Compositions and methods are provided for rapid characterization of Cas endonuclease systems and the elements comprising such systems, including, but not limiting to, rapid characterization of PAM sequences, guide RNA elements and Cas endonucleases. Type II Cas9 endonuclease systems originating from Brevibacillus laterosporus, Lactobacillus reuteri MIc3, 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.

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         7 . A recombinant DNA comprising at least one of randomized Protospacer-Adjacent-Motif (PAM) sequence adjacent to a target sequence that can be recognized by a guide RNA/Cas endonuclease complex. 
     
     
         8 . A method for identification of a tracrRNA of an organism, the method comprising:
 a) providing a first single guide RNA candidate comprising a chimeric non-naturally occurring crRNA comprising a variable targeting domain capable of hybridizing to a target sequence in the genome of a cell, linked to a first nucleotide sequence representing the sense expression of a candidate tracrRNA naturally occurring in said organism;   b) providing a second single guide RNA candidate comprising a chimeric non-naturally occurring crRNA comprising a variable targeting domain capable of hybridizing to a target sequence in the genome of said cell, linked to a second nucleotide sequence representing the sense expression of a candidate tracrRNA naturally occurring in said organism;   c) providing to the first and second single guide RNA candidates a Cas endonuclease protein, wherein said Cas endonuclease protein can form a complex with either the first single guide RNA candidate or the second single guide RNA candidate, wherein said complex is capable of introducing a double strand break into said target sequence; and,   d) identification of the first or second guide RNA candidate and its tracrRNA component that complexes to the Cas endonuclease of (c) and results in cleavage of the target sequence in the genome of said cell.   
     
     
         9 . A method for identification of a tracrRNA of an organism, the method comprising:
 a) identifying a CRISPR array repeat sequence in a genomic locus of said organism;   b) aligning the CRISPR array repeat sequence of (a) with the sequence of the genomic locus of (a) and identifying an antirepeat sequence that encodes a tracrRNA; and,   c) determining the transcriptional direction of the tracrRNA.   
     
     
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         15 . A method for modifying a target site in the genome of a cell, the method comprising providing to said cell at least one Cas9 endonuclease of SEQ ID NO: 140, or a functional fragment thereof, and at least one guide RNA, wherein said guide RNA and Cas endonuclease can form a complex that is capable of recognizing, binding to, and optionally nicking or cleaving all or part of said target site. 
     
     
         16 . The method of  claim 15 , further comprising identifying at least one cell that has a modification at said target, 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). 
     
     
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         20 . The method of  claim 15 , 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. 
     
     
         21 . The method of  claim 20 , wherein the plant cell is selected from the group consisting of a monocot and dicot cell. 
     
     
         22 . The method of  claim 21 , wherein the plant cell is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tomato, tobacco,  Arabidopsis , and safflower cell. 
     
     
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         25 . The recombinant DNA of  claim 7 , wherein the randomized PAM sequence comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 randomized nucleotides. 
     
     
         26 . The recombinant DNA of  claim 7 , wherein the target sequence is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. 
     
     
         27 . The recombinant DNA of  claim 7 , wherein the Cas endonuclease in the guide RNA/Cas endonuclease complex comprises a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3 protein, Cas3-HD protein, Cas 5 protein, Cas7 protein, Cas8 protein, or Cas10 protein. 
     
     
         28 . The recombinant DNA of  claim 7 , wherein the Cas endonuclease in the guide RNA/Cas endonuclease complex is a Cas9 endonuclease selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 and 140. 
     
     
         29 . The recombinant DNA of  claim 7 , wherein the guide in the guide RNA/Cas endonuclease complex is a single guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave a target sequence, wherein said single guide RNA is selected from the group consisting of SEQ ID NOS: 47 and 127. 
     
     
         30 . The recombinant DNA of  claim 7 , wherein the guide in the guide RNA/Cas endonuclease complex is a single guide RNA capable of forming a guide RNA/Cas9 endonuclease complex, wherein said guide RNA/Cas9 endonuclease complex can recognize, bind to, and optionally nick or cleave a target sequence, wherein said single guide RNA is selected from the group consisting of SEQ ID NOS: 47 and 127. 
     
     
         31 . The recombinant DNA of  claim 7 , wherein the guide in the guide RNA/Cas endonuclease complex is a guide RNA capable of forming a guide RNA/Cas endonuclease complex, wherein said guide RNA/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave a target sequence, wherein said guide RNA is a duplex molecule comprising a chimeric non-naturally occurring crRNA comprising SEQ ID NO: 185 and a tracrRNA comprising SEQ ID NO: 199, wherein said chimeric non-naturally occurring crRNA comprises a variable targeting domain capable of hybridizing to said target sequence. 
     
     
         32 . The recombinant DNA of  claim 7 , wherein the guide RNA/Cas endonuclease complex is a guide RNA/Cas endonuclease complex comprising a Cas9 endonuclease of SEQ ID NO: 140, or a functional fragment thereof, and a guide RNA, 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.