PLANT GENOME MODIFICATION USING GUIDE RNA/CAS ENDONUCLEASE SYSTEMS AND METHODS OF USE
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 obtaining a progeny plant comprising an altered target site in its plant genome, the method comprising:
a) crossing a first plant comprising at least one Cas endonuclease capable of introducing a double strand break at a target site in the plant genome with a second plant comprising a guide RNA capable of forming a complex with the Cas endonuclease of, to obtain progeny plants;
b) evaluating progeny plants of (a) for an alteration in the target site; and,
c) obtaining a progeny plant that possesses the desired alteration of said target site.
2 . (canceled)
3 . A method for obtaining a progeny plant comprising an altered target site in its plant genome, the method comprising:
a) crossing a first plant comprising at least one Cas endonuclease capable of introducing a double strand break at a target site in the plant genome with a second plant comprising a guide RNA and a donor DNA to obtain progeny plants, wherein said guide RNA is capable of forming a complex with the Cas endonuclease, wherein said donor DNA comprises a polynucleotide of interest;
b) evaluating progeny plants of (a) for an alteration in the target site;
and,
c) obtaining a progeny plant that comprises the polynucleotide of interest inserted at said target site.
4 . (canceled)
5 . 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 a Cas endonuclease, wherein said guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site.
6 . (canceled)
7 . The method of claim 5 , further comprising providing a donor DNA to said 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 SV40 nuclear targeting signal upstream of the Cas codon region and a VirD2 nuclear localization signal downstream of the Cas codon region.
15 . The method of claim 5 , wherein the plant is a monocot or a dicot.
16 . The method of claim 15 , wherein the monocot is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass.
17 . The method of claim 15 , wherein the dicot is selected from the group consisting of soybean, canola, alfalfa, sunflower, cotton, tobacco, tomato peanut, potato, tobacco, Arabidopsis , or safflower.
18 . (canceled)
19 . A plant or seed produced by the method of claim 5 .
20 . 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.
21 - 28 . (canceled)
29 . A method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing at least one guide RNA and at least one polynucleotide modification template into a cell comprising at least one Cas endonuclease, wherein the Cas endonuclease introduces a double-strand break at a target site in the genome of said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence.
30 . The method of claim 29 , wherein the cell is a plant cell.
31 . The method of claim 29 , wherein the nucleotide sequence in the genome of said cell is selected from the group consisting of a promoter sequence, a terminator sequence, a regulatory sequence, a splice site, a coding sequence, a polyubiquitination site, an intron site, an intron enhancing motif and a gene of interest.
32 . The method of claim 31 wherein the gene of interest is an enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene or an acetolactate synthase (ALS) gene.
33 . The method of claim 30 wherein the plant cell is a monocot or dicot plant cell.
34 - 36 . (canceled)
37 . A plant or seed produced from the plant cell of claim 30 .
38 . The method of claim 29 , wherein the Cas endonuclease is a Cas9 endonuclease.
39 - 42 . (canceled)
43 . The method of claim 29 , wherein the at least one nucleotide modification is not a modification at said target site.
44 - 66 . (canceled)