PRECISE INTRODUCTION OF DNA OR MUTATIONS INTO THE GENOME OF WHEAT
The present invention is in the field of genome editing and is directed to a method for the seam-less introduction of targeted precise modifications in genomic DNA of wheat.
1 . A method for precise introduction of at least one donor DNA molecule into a target region of the genome of wheat comprising the steps of
a. introducing into a wheat cell
i. at least one donor DNA molecule and
ii. at least one RNA guided nuclease or RNA guided nickase and
iii. at least one singleguideRNA (sgRNA) or tracrRNA and crRNA, and
b. incubating the wheat cell to allow for introduction of said at least one donor DNA into said target region of the genome and
c. selectin a wheat cell comprising the sequence of the donor DNA molecule in said target region,
wherein the donor DNA is functionally linked to at least 30 bases at its 5′ and/or 3′ end that are each at least 80% identical to a sequence in the target region.
2 . A method for producing a wheat plant comprising a donor DNA in a target region of the genome comprising the steps of
a. introducing into a wheat cell
i. at least one donor DNA and
ii. at least one RNA guided nuclease or RNA guided nickase and
iii. at least one single guideRNA (sgRNA) or tracrRNA and crRNA, and
b. incubating the wheat cell to allow for introduction of said at least one donor DNA into the target region in the genome
c. selectin a wheat cell comprising the sequence of the donor DNA molecule in said target region, and
d. regenerating a wheat plant from said selected wheat cell,
wherein the donor DNA is functionally linked to at least 30 bases at its 5′ and/or 3′ end that are each at least 80% identical to a sequence in the target region.
3 . The method of claim 1 , wherein after step b. the wheat cell is incubated on a medium comprising a selection agent.
4 . The method of claim 1 , wherein the RNA guided nuclease or the RNA guided nickase is a Cas nuclease or Cas nickase.
5 . The method of claim 1 , wherein the Cas nuclease or Cas nickase is a Cas9 or Cas12a nuclease or a Cas9 or Cas12a nickase.
6 . The method of claim 1 , wherein at least one of the at least one nuclease or at least one nickase or the at least one sgRNA or at least one crRNA and tracrRNA is introduced into said cell encoded by a nucleic acid molecule.
7 . The method of claim 6 , wherein the nucleic acid molecule is a plasmid comprising an expression cassette encoding said at least one nuclease/nickase or the at least one sgRNA or at least one crRNA and tracrRNA.
8 . The method of claim 6 , wherein the nucleic acid is an RNA molecule.
9 . The method of claim 6 , wherein the at least one nuclease or at least one nickase is sequence optimized for expression in wheat.
10 . The method of claim 1 , wherein the at least one RNA guided nuclease or RNA guided nickase and the at least one sgRNA or at least one crRNA and tracrRNA are introduced into said cell as ribonucleoprotein (RNP) assembled outside said cell.
11 . The method of claim 1 , wherein a combination of donor DNA and crRNA/tracrRNA or sgRNA is preselected for efficient introduction of the donor DNA molecule into the target region.
12 . The method of claim 1 , wherein the at least one donor DNA and at least one RNA guided nuclease or RNA guided nickase and at least one singleguideRNA (sgRNA) or tracrRNA and crRNA are introduced into said cell using particle bombardment or Agrobacterium mediated introduction of DNA.
13 . The method of claim 1 , wherein the at least one RNA guided nuclease or at least one RNA guided nickase is comprising a nuclear localization signal.
14 . The method of claim 2 , wherein after step b. the wheat cell is incubated on a medium comprising a selection agent.
15 . The method of claim 2 , wherein the RNA guided nuclease or the RNA guided nickase is a Cas nuclease or Cas nickase.
16 . The method of claim 2 , wherein the Cas nuclease or Cas nickase is a Cas9 or Cas12a nuclease or a Cas9 or Cas12a nickase.
17 . The method of claim 2 , wherein at least one of the at least one nuclease or at least one nickase or the at least one sgRNA or at least one crRNA and tracrRNA is introduced into said cell encoded by a nucleic acid molecule.
18 . The method of claim 17 , wherein the nucleic acid molecule is a plasmid comprising an expression cassette encoding said at least one nuclease/nickase or the at least one sgRNA or at least one crRNA and tracrRNA.
19 . The method of claim 17 , wherein the nucleic acid is an RNA molecule.
20 . The method of claim 17 , wherein the at least one nuclease or at least one nickase is sequence optimized for expression in wheat.
21 . The method of claim 2 , wherein the at least one RNA guided nuclease or RNA guided nickase and the at least one sgRNA or at least one crRNA and tracrRNA are introduced into said cell as ribonucleoprotein (RNP) assembled outside said cell.
22 . The method of claim 2 , wherein a combination of donor DNA and crRNA/tracrRNA or sgRNA is preselected for efficient introduction of the donor DNA molecule into the target region.
23 . The method of claim 2 , wherein the at least one donor DNA and at least one RNA guided nuclease or RNA guided nickase and at least one singleguideRNA (sgRNA) or tracrRNA and crRNA are introduced into said cell using particle bombardment or Agrobacterium mediated introduction of DNA.
24 . The method of claim 2 , wherein the at least one RNA guided nuclease or at least one RNA guided nickase is comprising a nuclear localization signal.