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 .- 66 . (canceled) 
     
     
         67 . A method for modifying a target site in the genome of a plant cell, the method comprising providing a guide RNA to a plant cell having stably integrated into its genome a Cas9 endonuclease gene, wherein said guide RNA and the Cas9 endonuclease encoded by said gene are capable of forming a complex that enables the Cas9 endonuclease to introduce a double-strand break at said target site. 
     
     
         68 . The method of  claim 67 , wherein the Cas9 endonuclease gene is a plant-optimized Cas9 endonuclease. 
     
     
         69 . The method of  claim 67 , further comprising providing a donor DNA to said plant cell, wherein said donor DNA comprises a polynucleotide of interest. 
     
     
         70 . The method of  claim 69 , further comprising identifying at least one modified plant cell comprising in its genome the polynucleotide of interest integrated at said target site. 
     
     
         71 . The method of  claim 67 , wherein said guide RNA is introduced directly by particle bombardment. 
     
     
         72 . The method of  claim 67 , wherein the Cas9 endonuclease gene is operably linked to a SV40 nuclear targeting signal upstream of the Cas9 codon region and a VirD2 nuclear localization signal downstream of the Cas9 codon region. 
     
     
         73 . The method of  claim 67 , wherein the plant is a monocot or a dicot. 
     
     
         74 . The method of  claim 73 , wherein the monocot is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, and switchgrass. 
     
     
         75 . The method of  claim 73 , wherein the dicot is selected from the group consisting of soybean, canola, alfalfa, sunflower, cotton, tobacco, tomato, peanut, potato,  Arabidopsis , and safflower. 
     
     
         76 . A plant comprising a recombinant DNA construct, said recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence encoding a plant-optimized Cas9 endonuclease, wherein said plant-optimized Cas9 endonuclease is capable of binding to and creating a double-strand break in a genomic target sequence of said plant genome, and wherein said plant does not comprise a guide RNA. 
     
     
         77 . A method for editing a nucleotide sequence in the genome of a plant cell, the method comprising introducing at least one guide RNA and at least one polynucleotide modification template into a plant cell having stably integrated into its genome a Cas9 endonuclease gene, wherein the Cas9 endonuclease encoded by said gene is capable of introducing a double-strand break at a target site in the genome of said plant cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence. 
     
     
         78 . The method of  claim 77 , wherein the nucleotide sequence in the genome of said plant cell is selected from the group consisting of a promoter, a regulatory sequence, a splice site, a coding sequence, a polyubiquitination site, an intron site, an intron enhancing motif and a gene of interest, optionally wherein the gene of interest is an enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene or an acetolactate synthase (ALS) gene. 
     
     
         79 . The method of  claim 77 , wherein the plant cell is a monocot plant cell or a dicot plant cell. 
     
     
         80 . The method of any one of  claim 77 , wherein the at least one nucleotide modification is not a modification at said target site. 
     
     
         81 . A transgenic plant comprising a Cas9 endonuclease gene stably integrated into its genome, wherein the Cas9 endonuclease gene is present in the absence of a guide RNA.