Method for conducting site-specific modification on entire plant via gene transient expression

Abstract

The present invention discloses a method for site-directed modification of whole plant through gene transient expression. The method as provided for conducting site-directed modification to a target fragment of a target gene in a whole plant comprises the following steps: transiently expressing a sequence-specific nuclease in said plant, wherein a whole plant is used as the subject for transient expression, said sequence-specific nuclease targets and cleaves said target fragment, thereby the site-directed modification is achieved via the self DNA repairing of said plant. In the present invention, tissue culture is omitted by transient expression of the sequence-specific nuclease; mutation is obtained at whole plant level; the method is independent of the genotype and recipient, and thus can be applied to various varieties of various species; T1 mutants can be obtained directly and the mutation can be stable inherited; more importantly, the mutant plant as obtained is free of exogenous genes, and thus have higher bio-safety.

1 - 16 . (canceled) 
     
     
         17 . A method for conducting site-directed modification to a target fragment of a target gene in a whole plant, comprising:
 transiently expressing a sequence-specific nuclease in said plant, wherein a whole plant is used as the subject for transient expression, said sequence-specific nuclease targets and cleaves said target fragment, whereby the site-directed modification is achieved via the self DNA repairing of said plant, wherein the method for transiently expressing said site-directed nuclease in said plant comprises the following steps:   a) delivering the sequence-specific nuclease or a genetic material for expressing the sequence-specific nuclease into a leaf of said plant, and   b) growing the plant obtained in step a) in the absence of selection pressure, whereby the sequence-specific nuclease or the genetic material not integrated into the plant chromosome is degraded,   wherein step b) does not comprise tissue culture,   wherein said genetic material is a recombinant vector or a DNA linear fragment or an in vitro transcribed RNA;   wherein when said part of plant is a leaf, the delivery is performed by injecting a solution of  Agrobacterium tumefaciens  carrying said recombinant vector or DNA linear fragment into the leaf.   
     
     
         18 . The method according to  claim 17 ,
 wherein the delivery is performed by injecting a solution of  Agrobacterium tumefaciens  carrying said recombinant vector or DNA linear fragment into the leaf.   
     
     
         19 . The method of  claim 17 , wherein said sequence-specific nuclease is a CRISPR/Cas9 nuclease, a TALENs nuclease, a Zinc finger nuclease, or any nuclease that can achieve genome editing,
 wherein when the sequence-specific nuclease is a CRISPR/Cas9 nuclease, the genetic material is composed of   a recombinant vector or DNA fragment capable of transcribing guide RNA and expressing Cas9 protein;   a recombinant vector or DNA fragment capable of transcribing guide RNA and a recombinant vector or DNA fragment or RNA capable of expressing Cas9 protein; or a guide RNA and a recombinant vector or DNA fragment or RNA capable of expressing Cas9 protein,   wherein the guide RNA is an RNA with a palindromic structure which is formed by partial base-pairing between crRNA and tracrRNA and the crRNA contains an RNA fragment that can complementarily bind to the target fragment;   wherein when the sequence-specific nuclease is a TALENs nuclease, the genetic material is a recombinant vector or DNA fragment or RNA capable of expressing paired TALEN proteins;   wherein the TALEN protein is composed of a DNA binding domain capable of recognizing and binding to the target site, and a Fok I domain;   wherein when the sequence-specific nuclease is a Zinc finger nuclease, the genetic material is a recombinant vector DNA fragment or RNA capable of expressing paired ZFN proteins; and   wherein the ZFN protein is composed of a DNA binding domain capable of recognizing and binding to the target site, and a Fok I domain.   
     
     
         20 . The method of  claim 17 , wherein the site-directed modification is an insertion, deletion, and/or replacement mutation in the target fragment. 
     
     
         21 . A method for making a transgene-free mutant plant comprising performing site-directed modification to a target fragment of a target gene in a plant of interest according to the method of  claim 17  so as to obtain a plant in which a function of the target gene is lost and the genome of the plant is free of integrated exogenous genes. 
     
     
         22 . The method of  claim 17 , wherein said plant is a plant of any genotype. 
     
     
         23 . The method of  claim 17 , wherein the plant is selected from the group consisting of tobacco,  Brassica oleracea, Fagopyrum tataricum , or  Hevea brasiliensis.