Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof

Abstract

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

1 - 57 . (canceled) 
     
     
         58 . A method of cleaving a target DNA in a plant cell, wherein the target DNA is endogenous DNA, the method comprising introducing into the plant cell a CRISPR/Cas9 complex comprising:
 (a) a CRISPR-associated protein 9 (Cas9) polypeptide; and   (b) a guide RNA specific to the target DNA,   wherein the guide RNA is:   (i) a dual guide RNA comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA); or   (ii) a single-chain guide RNA comprising a CRISPR RNA (crRNA) fused to a trans-activating crRNA (tracrRNA);   
       whereby the target DNA in the plant cell is cleaved. 
     
     
         59 . A method of introducing two nicks in different strands of a target DNA in a plant cell, wherein the target DNA is endogenous DNA, the method comprising introducing into the plant cell a CRISPR/Cas9 system comprising:
 (a) a CRISPR-associated protein 9 (Cas9) polypeptide, wherein the Cas9 polypeptide is a Cas9 nickase; and   (b) two guide RNAs, wherein each of the two guide RNAs is specific for one of the strands of the target DNA,   wherein the guide RNA is:   (i) a dual guide RNA comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA); or   (ii) a single-chain guide RNA comprising a CRISPR RNA (crRNA) fused to a trans-activating crRNA (tracrRNA);   
       wherein each of the two guide RNAs forms a complex with the Cas9 polypeptide; and 
       whereby two nicks are introduced in the target DNA. 
     
     
         60 . The method of  claim 59 , wherein the two nicks are separated by at least 100 base pairs. 
     
     
         61 . The method of  claim 58 , wherein the CRISPR/Cas9 complex is introduced by transfection, wherein the transfection is performed by the method selected from the group consisting of microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, virus-mediated gene delivery, or polyethylene glycol (PEG)-mediated transfection of protoplasts. 
     
     
         62 . The method of  claim 61  wherein the transfection is by PEG-mediated transfection of protoplasts in a 40% PEG transfection buffer. 
     
     
         63 . The method of  claim 62 , wherein the 40% PEG transfection buffer comprises 40% PEG4000, 200 mM mannitol and 100 mM CaCl 2 . 
     
     
         64 . The method of  claim 58 , wherein the Cas9 protein is from the genus  Streptococcus.    
     
     
         65 . The method of  claim 64  or  claim 63 , wherein the Cas9 protein is from  Streptococcus pyogenes.    
     
     
         66 . The method of  claim 59 , wherein the Cas9 nickase is a mutant form of Cas9, in which the catalytic aspartate residue is changed to alanine. 
     
     
         67 . The method of  claim 58 , wherein the Cas9 polypeptide comprises a nuclear localization signal (NLS). 
     
     
         68 . The method of  claim 58 , wherein the crRNA comprises two additional nucleotides at its 5′ end, wherein the two additional nucleotides are guanine nucleotides. 
     
     
         69 . The method of  claim 58 , wherein the crRNA is 20 nucleotides in length. 
     
     
         70 . The method of  claim 58 , wherein the target DNA is genomic DNA. 
     
     
         71 . The method of  claim 58 , wherein the target DNA comprises a trinucleotide protospacer adjacent motif (PAM) recognized by Cas9, wherein the PAM consists of the trinucleotide 5′-NGG-3′. 
     
     
         72 . The method of  claim 58  or  59 , wherein the plant cell is a protoplast. 
     
     
         73 . A plant cell comprising a target DNA, wherein the target DNA has been modified using the method of  claim 58  or  59 . 
     
     
         74 . A plant comprising the plant cell of  claim 73 .