Plant genome modification using guide rna/cas endonuclease systems and methods of use

Abstract

Compositions and methods are provided for genome modification of a target sequence in the genome of a plant or plant cell. The methods and compositions employ a guide RNA/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. Also provided are compositions and methods employing a guide polynucleotide/Cas endonuclease system for genome modification of a nucleotide sequence in the genome of a cell or organism, for gene editing, and/or for inserting or deleting a polynucleotide of interest into or from the genome of a cell or organism. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Breeding methods and methods for selecting plants utilizing a two component RNA guide and Cas endonuclease system are also disclosed. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell.

1 . A method for cultivating a plant cell, the method comprising:
 (a) transforming at least one plant cell with a guide polynucleotide and a polynucleotide sequence encoding a polynucleotide-guided endonuclease, wherein the guide polynucleotide and the polynucleotide-guided endonuclease form a complex that introduces a double-strand break at the target site in the plant cell;   (b) providing an increased level of activity of one or more polypeptides encoded by one or more developmental genes in the plant cell; and   (c) regenerating a plantlet or a plant from the plant cell, the plantlet or plant comprising a modified target site, wherein the plantlet or the plant does not contain the polynucleotide-guided endonuclease or the increased level of activity of the one or more developmental genes.   
     
     
         2 . The method of  claim 1 , wherein the at least one plant cell is an embryogenic plant cell. 
     
     
         3 . The method of  claim 2 , wherein the embryogenic plant cell is transformed by particle bombardment. 
     
     
         4 . The method of  claim 2 , wherein the at least one plant cell comprises a plurality of embryogenic plant cells and the double-strand break at the target site is induced in the plurality of embryogenic plant cells at a frequency of at least 10.1%. 
     
     
         5 . The method of  claim 1 , wherein the at least one plant cell is an embryo. 
     
     
         6 . The method of  claim 5 , wherein the embryo is transformed by particle bombardment or bacterial-mediated transformation. 
     
     
         7 . The method of  claim 5 , wherein the at least one plant cell comprises a plurality of embryos and the double-strand break at the target site is induced in the plurality of embryos at a frequency of at least 33%. 
     
     
         8 . The method of  claim 1 , wherein the modified target site comprises at least one nucleotide insertion, deletion, or substitution. 
     
     
         9 . The method of  claim 1 , wherein the one or more developmental genes is a BBM gene or a WUS gene. 
     
     
         10 . The method of  claim 1 , wherein the modified target site is a promoter sequence, a terminator sequence, a regulatory element sequence, a splice site, a coding sequence, a polyubiquitination site, an intron site, an intron-enhancing motif, or a gene of interest. 
     
     
         11 . The method of  claim 1 , wherein the plant cell is monocot or dicot. 
     
     
         12 . The method of  claim 11 , wherein the monocot is maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass. 
     
     
         13 . The method of  claim 11 , wherein the dicot is soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, Arabidopsis, or safflower. 
     
     
         14 . The method of  claim 1 , further comprising transforming the at least one plant cell with a polynucleotide modification template comprising at least one nucleotide modification relative to a polynucleotide sequence of the target site. 
     
     
         15 . The method of  claim 1 , wherein the polynucleotide-guided endonuclease is a Cas endonuclease and the polynucleotide sequence encoding the polynucleotide-guided endonuclease comprises a Cas coding region that is operably linked to a first nuclear localization signal upstream of the Cas coding region and a second nuclear localization signal downstream of the Cas coding region, wherein the first nuclear localization signal and the second nuclear localization signal are derived from different sources. 
     
     
         16 . The method of  claim 15 , wherein the first nuclear localization signal is a monopartite nuclear localization signal. 
     
     
         17 . The method of  claim 15 , wherein the second nuclear localization signal is a bipartite nuclear localization signal. 
     
     
         18 . The method of  claim 1 , wherein the plant cell is a non-protoplast plant cell. 
     
     
         19 . The method of  claim 1 , wherein providing an increased level of activity of one or more polypeptides encoded by one or more developmental genes comprises transforming the plant cell with one or more expression cassettes expressing the one or more developmental genes. 
     
     
         20 . The method of  claim 19 , wherein the plant cell is transformed with the one or more expression cassettes by particle bombardment or bacterial-mediated transformation. 
     
     
         21 . The method of  claim 1 , wherein providing the increased level of activity of the one or more polypeptides encoded by the one or more developmental genes enhances genome editing of the plant cell.