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 - 4 . (canceled) 
     
     
         5 . A method for modifying a target site in the genome of a plant cell, the method comprising:
 a. providing a guide RNA to a plant cell, wherein said guide RNA forms a complex with a Cas endonuclease to introduce a double strand break at said target site; and   b. identifying at least nucleotide modification at the target site.   
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 5 , further comprising providing a donor DNA to the plant cell, wherein said donor DNA comprises a polynucleotide of interest. 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The method of  claim 5 , wherein the guide RNA is introduced directly by particle bombardment. 
     
     
         12 . The method of  claim 5 , wherein the guide RNA is introduced via particle bombardment or  Agrobacterium  transformation of a recombinant DNA construct comprising the corresponding guide DNA operably linked to a plant U6 polymerase III promoter. 
     
     
         13 . The method of  claim 5 , wherein the Cas endonuclease gene is a plant-optimized Cas9 endonuclease. 
     
     
         14 . The method of  claim 5 , wherein the Cas endonuclease gene is operably linked to a nuclear targeting signal upstream of the Cas codon region and a nuclear localization signal downstream of the Cas codon region. 
     
     
         15 . The method of  claim 5 , wherein the plant cell is obtained from a monocot plant or a dicot plant. 
     
     
         16 . The method of  claim 15 , wherein the monocot plant is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass. 
     
     
         17 . The method of  claim 16 , wherein the dicot plant is selected from the group consisting of soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, Arabidopsis, or safflower. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 - 66 . (canceled)