Method for producing genome-modified plants from plant protoplasts at high efficiency

Abstract

The present invention relates to a method of increasing the production efficiency of gene-edited plants, regenerated from plant protoplasts, by use of a Cas protein-guide RNA ribonucleoprotein (RNP). According to the present invention, the method of increasing the production efficiency of gene-edited plants makes it possible to efficiently produce target gene-mutated plants and to minimize the introduction of foreign DNA into plants. Thus, the present invention can be very advantageously used in a wide variety of fields, including agriculture, food and biotechnology.

1 . A method for increasing the production efficiency of a genome-edited plant from a plant protoplast, comprising the steps of:
 (i) editing a genome of the plant protoplast by a direct introduction of a Cas protein-guide RNA ribonucleoprotein (RNP) in which a Cas protein and a guide RNA in the form of naked RNA are pre-assembled into an isolated plant protoplast without using a vector; and   (ii) producing a genome-edited plant by regenerating the plant protoplast.   
     
     
         2 . The method of  claim 1 , wherein the guide RNA is specific for a DNA encoding a target gene. 
     
     
         3 . The method of  claim 2 , wherein the target gene is a Brassinosteroid Insensitive 2 (BIN2) gene or a Glucosinolate-oxoglutarate-dependent dioxygenase homolog (GSL-ALK) gene. 
     
     
         4 . The method of  claim 1 , wherein the editing of a genome is performed by knocking-out or knocking-in. 
     
     
         5 . The method of  claim 1 , wherein the guide RNA is in the form of a dual RNA comprising a crRNA and a tracrRNA, or a single-chain guide RNA (sgRNA). 
     
     
         6 . The method of  claim 5 , wherein the single-chain guide RNA comprises a part of crRNA and a part of tracrRNA. 
     
     
         7 . The method of  claim 1 , wherein the Cas protein is a Cas9 protein or a variant of Cas9 Protein in which the catalytic aspartate residue is substituted with another amino acid. 
     
     
         8 . The method of  claim 1 , wherein the Cas protein recognizes NGG trinucleotide. 
     
     
         9 . The method of  claim 1 , wherein the Cas protein is linked to a protein transduction domain. 
     
     
         10 . The method of  claim 7 , wherein the amino acid is alanine. 
     
     
         11 . The method of  claim 1 , wherein the Cas9 protein is derived from the genus  Streptococcus.    
     
     
         12 . The method of  claim 11 , wherein the genus  Streptococcus  is  Streptococcus pyogenes.    
     
     
         13 . The method of  claim 1 , wherein the plant protoplast is derived from  Lactuca sativa  or  Brassica oleracea.    
     
     
         14 . The method of  claim 1 , wherein the introduction 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, and PEG-mediated transfection. 
     
     
         15 . A plant regenerated from the genome-edited plant protoplast produced by the method of  claim 1 .