Method for base editing in plants

Abstract

The present invention belongs to the field of plant genetic engineering. Specifically, the invention relates to a method for base editing in plants. More particularly, the invention relates to a method for performing efficient base editing to a target sequence in the genome of a plant (such as a crop plant) by a Cas9-cytidine deaminase fusion protein, as well as plants produced through said method and progenies thereof.

1 - 23 . (canceled) 
     
     
         24 . A method for producing a genetically modified plant or plant cell, comprising introducing in the absence of any selective pressure into the plant or plant cell a composition for performing base editing to a target sequence in a plant genome, comprising at least one of the following (i) to (v):
 i) a base editing fusion protein, and a guide RNA;   ii) an expression construct comprising a nucleotide sequence encoding a base editing fusion protein, and a guide RNA;   iii) a base editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;   iv) an expression construct comprising a nucleotide sequence encoding a base editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;   v) an expression construct comprising a nucleotide sequence encoding base editing fusion protein and a nucleotide sequence encoding guide RNA;   wherein said base editing fusion protein contains a nuclease-inactivated Cas9 domain and a deaminase domain, said guide RNA can target said base editing fusion protein to the target sequence in the plant genome,   and thereby said guide RNA target said base editing fusion protein to a target sequence in the genome of said plant or plant cell, resulting in one or more C to T substitutions in said target sequence,   wherein said deaminase is an APOBEC1 deaminase and said nuclease-inactivated Cas9 comprises amino acid substitutions of D10A and/or H840A relative to wild-type Cas9.   
     
     
         25 . The method according to  claim 24 , wherein one or more C in the positions 3-9 of the protospacer sequence in said target sequence is substituted with T. 
     
     
         26 . The method according to  claim 24 , further comprising screening for plants or plant cells with desired nucleotide substitution(s). 
     
     
         27 . The method according to  claim 24 , wherein said plant or plant cell is selected from monocotyledon and dicotyledon. 
     
     
         28 . The method according to  claim 27 , wherein said plant is a crop plant, such as wheat, rice, maize, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugar cane, tomato, tobacco, cassava, or potato. 
     
     
         29 . The method according to  claim 24 , wherein said target sequence is associated with a plant trait such as an agronomy trait, and thereby said base editing results in a change of the trait in the plant relative to a wild-type plant. 
     
     
         30 . The method according to  claim 24 , wherein said composition is introduced into said plant or plant cell through a method selected from particle bombardment, PEG-mediated protoplast transformation,  Agrobacterium -mediated transformation, plant virus-mediated transformation, a pollen tube approach, and ovary injection approach. 
     
     
         31 . The method according to  claim 24 , further comprising obtaining a progeny of the genetically modified plant. 
     
     
         32 . A genetically modified plant or a progeny thereof or a part thereof, wherein said plant is obtained through the method according to  claim 24 . 
     
     
         33 . A plant breeding method, comprising crossing a first plant genetically modified through the method according to  claim 24  with a second plant that does not contain said genetic modification, and thereby introducing said genetic modification into said second plant. 
     
     
         34 . The method according to  claim 24 , wherein said deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase having a deamination window of 7 nucleotides at positions 3-9 of the protospacer sequence 
     
     
         35 . The method according to  claim 34 , wherein said APOBEC1 deaminase comprises an amino acid sequence set forth in SEQ ID NO: 11. 
     
     
         36 . The method according to  claim 24 , wherein said nuclease-inactivated Cas9 comprises an amino acid sequence set forth in SEQ ID NO:13 or 14. 
     
     
         37 . The method according to  claim 24 , wherein said deaminase domain is fused to the N-terminal of said nuclease-inactivated Cas9 domain, or wherein said deaminase domain is fused to the C-terminal of said nuclease-inactivated Cas9 domain. 
     
     
         38 . The method according to  claim 24 , wherein said deaminase domain and said nuclease inactivated Cas9 domain is fused through a linker, for example said linker is XTEN linker set forth in SEQ ID NO: 12. 
     
     
         39 . The method according to  claim 24 , wherein said base editing fusion protein further comprises an uracil DNA glycosylase inhibitor (UGI), for example said uracil DNA glycosylase inhibitor comprises an amino acid sequence set forth in SEQ ID NO: 15. 
     
     
         40 . The method according to  claim 24 , wherein said base editing fusion protein further comprises a nuclear localization sequence (NLS), for example said NLS comprises an amino acid sequence set forth in SEQ ID NO: 30 or 31. 
     
     
         41 . The method according to  claim 24 , wherein the base editing fusion protein has two NLSs, and the two NLSs are located at the N-terminus and C-terminus, respectively. 
     
     
         42 . The method according to  claim 24 , wherein said base editing fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 22 or 23. 
     
     
         43 . The method according to  claim 24 , wherein the nucleotide sequence encoding said base editing fusion protein is codon optimized for plants to be base edited, for example, said nucleotide sequence encoding said base editing fusion protein is set forth in SEQ ID NO: 19 or 20. 
     
     
         44 . The method according to  claim 24 , wherein said guide RNA is a single guide RNA (sgRNA). 
     
     
         45 . The method according to  claim 24 , wherein the nucleotide sequence encoding said base editing fusion protein and/or the nucleotide sequence encoding said guide RNA are operably linked to an expression regulatory element for the plant. 
     
     
         46 . The method according to  claim 45 , wherein said expression regulatory element is a promoter, for example a 35S promoter, a maize Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, or a maize U3 promoter.