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 maize cell, the method comprising:
 a) providing to the maize cell a guide RNA and a Cas9 endonuclease, wherein said guide RNA and Cas9 endonuclease form a complex that enables the Cas9 endonuclease to introduce a double strand break at the target site; and,   b) identifying at least one maize cell that has a modification of the target site, wherein the modification is selected from the group consisting of: at least one nucleotide insertion, at least one nucleotide deletion, at least one nucleotide substitution, and any combination of the preceding.   
     
     
         68 . The method of  claim 67 , wherein the guide RNA is introduced as a polyribonucleotide by particle bombardment. 
     
     
         69 . The method of  claim 67 , 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 promoter. 
     
     
         70 . The method of  claim 67 , wherein the Cas9 endonuclease is provided as a cas9 polynucleotide encoding a Cas9 polypeptide. 
     
     
         71 . The method of  claim 67 , wherein the Cas9 endonuclease is plant-optimized. 
     
     
         72 . The method of  claim 67 , wherein the Cas9 endonuclease is operably linked to a nuclear targeting signal and a nuclear localization signal. 
     
     
         73 . The method of  claim 67 , wherein the target site is selected from the group consisting of: a promoter sequence, a terminator sequence, a regulatory element sequence, a splice site, a coding sequence, a polyubiquitination site, an intron site, and an intron-enhancing motif. 
     
     
         74 . The method of  claim 67 , further comprising providing to the maize cell a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification as compared to the polynucleotide sequence of the target site. 
     
     
         75 . The method of  claim 67 , further comprising providing to the maize cell a donor DNA, wherein the donor DNA comprises a polynucleotide of interest or a promoter. 
     
     
         76 . A method for modifying a target site in the genome of a soybean plant cell, the method comprising:
 a) providing to the soybean cell a guide RNA and a Cas9 endonuclease, wherein said guide RNA and Cas9 endonuclease form a complex that enables the Cas9 endonuclease to introduce a double strand break at the target site; and,   b) identifying at least one soybean cell that has a modification of the target site, wherein the modification is selected from the group consisting of: at least one nucleotide insertion, at least one nucleotide deletion, at least one nucleotide substitution, and any combination of the preceding.   
     
     
         77 . The method of  claim 76 , wherein the guide RNA is introduced as a polyribonucleotide by particle bombardment. 
     
     
         78 . The method of  claim 76 , 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 promoter. 
     
     
         79 . The method of  claim 76 , wherein the Cas9 endonuclease is provided as a cas9 polynucleotide encoding a Cas9 polypeptide 
     
     
         80 . The method of  claim 76 , wherein the Cas9 endonuclease is plant-optimized. 
     
     
         81 . The method of  claim 76 , wherein the Cas9 endonuclease is operably linked to a nuclear targeting signal and a nuclear localization signal. 
     
     
         82 . The method of  claim 76 , wherein the target site is selected from the group consisting of: a promoter sequence, a terminator sequence, a regulatory element sequence, a splice site, a coding sequence, a polyubiquitination site, an intron site, and an intron-enhancing motif. 
     
     
         83 . The method of  claim 76 , further comprising providing to the soybean cell a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification as compared to the polynucleotide sequence of the target site. 
     
     
         84 . The method of  claim 76 , further comprising providing to the soybean cell a donor DNA, wherein the donor DNA comprises a polynucleotide of interest or a promoter. 
     
     
         85 . A maize cell comprising a Cas9 endonuclease and a guide RNA, wherein the Cas9 endonuclease and guide RNA form a complex that recognizes, binds to, and optionally creates a double strand break in a target sequence in the genome of the cell. 
     
     
         86 . The maize cell of  claim 85 , wherein the Cas9 endonuclease is plant-optimized. 
     
     
         87 . The maize cell of  claim 85 , wherein the guide RNA is present as a polyribonucleotide. 
     
     
         88 . The maize cell of  claim 85 , wherein the guide RNA is present as a polynucleotide encoding a polyribonucleotide. 
     
     
         89 . The maize cell of  claim 85 , wherein the Cas9 endonuclease is present as a polypeptide. 
     
     
         90 . The maize cell of  claim 85 , wherein the Cas9 endonuclease is present as a polynucleotide encoding a Cas9 polypeptide. 
     
     
         91 . A soybean cell comprising a Cas9 endonuclease and a guide RNA, wherein the Cas9 endonuclease and guide RNA form a complex that recognizes, binds to, and optionally creates a double strand break in a target sequence in the genome of the cell. 
     
     
         92 . The soybean cell of  claim 91 , wherein the Cas9 endonuclease is plant-optimized. 
     
     
         93 . The soybean cell of  claim 91 , wherein the guide RNA is present as a polyribonucleotide. 
     
     
         94 . The soybean cell of  claim 91 , wherein the guide RNA is present as a polynucleotide encoding a polyribonucleotide. 
     
     
         95 . The soybean cell of  claim 91 , wherein the Cas9 endonuclease is present as a polypeptide. 
     
     
         96 . The soybean cell of  claim 91 , wherein the Cas9 endonuclease is present as a polynucleotide encoding a Cas9 polypeptide.