Genome editing using campylobacter jejuni crispr/cas system-derived rgen

Abstract

The disclosure provided herewith relates to a Campylobacter jejuni CRISPR/CAS system-derived RGEN and a use thereof.

1 - 67  (canceled) 
     
     
         68 . A method of altering a genome via targeting a DNA sequence comprising a protospacer adjacent motif (PAM) sequence of NNNNRYAC (SEQ ID NO:1), the method comprising:
 introducing a Cas protein that recognizes the PAM sequence of SEQ ID NO:1, or a nucleic acid encoding the Cas protein, and;   an isolated guide RNA, comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence adjacent to a proto-spacer-adjacent motif (PAM) sequence of NNNNRYAC (SEQ ID NO: 1), or a nucleic acid encoding the isolated guide RNA into a cell comprising the genome, thereby altering the genome.   
     
     
         69 . The method of  claim 68 , wherein the Cas protein originates from a microorganism belonging to  Campylobacter.    
     
     
         70 . The method of  claim 69 , wherein the microorganism is  Campylobacter jejuni.    
     
     
         71 . The method of  claim 68 , wherein the Cas protein is a Cas9 protein. 
     
     
         72 . The method of  claim 68 , introducing a guide RNA is carried out simultaneously or sequentially with the Cas protein that recognizes the PAM sequence of SEQ ID NO: 1. 
     
     
         73 . The method of  claim 68 , wherein the guide RNA is a dual RNA comprising CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). 
     
     
         74 . The method of  claim 68 , wherein the guide RNA is a single guide RNA (sgRNA). 
     
     
         75 . The method of  claim 74 , wherein the sgRNA comprises a first region containing a sequence capable of forming a duplex with a complementary strand of the target DNA sequence, and a second region containing a sequence that interacts with the Cas protein. 
     
     
         76 . The method of  claim 74 , wherein the sgRNA comprises a portion of a crRNA which contains a sequence capable of forming a duplex with a complementary strand of the target DNA sequence, and a portion of a tracrRNA which contains a sequence interacting with the Cas protein. 
     
     
         77 . The method of  claim 68 , wherein the Cas protein having the nickase activity has a catalytic aspartic acid (D) at position 8 and histidine (H) at position 559 substituted with another amino acid. 
     
     
         78 . The method of  claim 77 , wherein the substituted amino acid is alanine. 
     
     
         79 . The method of  claim 68 , wherein the Cas protein binds to the target DNA sequence comprising the PAM sequence of SEQ ID NO: 1, without cleaving the target DNA. 
     
     
         80 . A composition for altering a genome with a target DNA sequence, comprising: an isolated guide RNA or a DNA encoding the isolated guide RNA, and a Cas9 protein that recognizes a PAM sequence of NNNNRYAC (SEQ ID NO:1) or a nucleic acid encoding the Cas9 protein, wherein the target DNA sequence is adjacent to the PAM sequence of NNNNRYAC (SEQ ID NO:1). 
     
     
         81 . The composition of  claim 80 , wherein the Cas protein is a deactivated Cas protein (dCas) or a nucleic acid that encodes the dCas. 
     
     
         82 . The composition of  claim 80 , wherein the isolated guide RNA is a dual RNA comprising a crRNA (CRISPR RNA) and a tracrRNA (trans-activating crRNA). 
     
     
         83 . The composition of  claim 80 , wherein the isolated guide RNA is a single-stranded guide RNA (sgRNA). 
     
     
         84 . The composition of  claim 83 , wherein the sgRNA comprises a first region containing a sequence capable of forming a duplex with a complementary strand of the target DNA sequence, and a second region containing a sequence interacting with the Cas protein. 
     
     
         85 . The composition of  claim 83 , wherein the length of the sequence capable of forming a duplex with a complementary strand of a target DNA sequence is 17 to 23 bp. 
     
     
         86 . The composition of  claim 83  wherein the guide RNA further comprises one to three additional nucleotides prior to a 5′ end of the sequence capable of forming a duplex with a complementary strand of the target DNA. 
     
     
         87 . The composition of  claim 86 , wherein the additional nucleotides comprise guanine (G).