GENE TARGETING IN PLANTS USING DNA VIRUSES

• US 20150225734A1
• Filed: 06/19/2013
• Published: 08/13/2015
• Est. Priority Date: 06/19/2012
• Status: Abandoned Application

• Current Assignee: Regents of The University of Minnesota (University of Minnesota)
• Original Assignee: Regents of The University of Minnesota (University of Minnesota)
• Inventors: Voytas, Daniel F., Baltes, Nicholas
• Application Number:
  US14/409,148
• Publication Number:
  US 20150225734A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/8203 Virus mediated transformation
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2750/12043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2999/007 Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Systems and methods for gene targeting in plants, including systems and methods that include the use of geminiviruses and customizable endonucleases.

Claims

1. A method for modifying the genetic material of a plant cell, comprising:

(a) introducing into the cell a virus nucleic acid comprising a donor sequence that is heterologous to the virus and is targeted to a first sequence that is endogenous to the plant cell; and

(b) inducing a double strand break at or near the sequence to which the donor sequence is targeted, wherein said double strand break is generated by an endonuclease targeted to a second endogenous plant sequence at or near the first sequence that is targeted by the donor sequence,

wherein homologous recombination occurs between the first endogenous plant sequence and the donor sequence.

2. The method of claim 1, wherein the virus nucleic acid is a plant DNA virus nucleic acid.

3. The method of claim 1, wherein the virus nucleic acid is a geminivirus nucleic acid.

4. The method of claim 1, wherein the endonuclease is a zinc finger nuclease, a transcription activator-like effector nuclease, a meganuclease, or a CRISPR/Cas system endonuclease.

5. The method of claim 1, wherein the endonuclease is encoded by a transgene sequence stably integrated into the genetic material of the plant, or is expressed transiently.

6. The method of claim 5, wherein the transgene encoding the endonuclease is operably linked to a promoter that is constitutive, cell specific, inducible, or activated by alternative splicing of a suicide exon.

7. The method of claim 1, wherein the virus nucleic acid comprises a sequence encoding the endonuclease.

8. The method of claim 1, further comprising introducing into the plant cell an RNA virus nucleic acid comprising a nucleotide sequence encoding the endonuclease.

9. The method of claim 8, wherein the RNA virus nucleic acid is introduced into the plant cell after or simultaneous with step (a).

10. The method of claim 8, wherein the RNA virus nucleic acid is from a tobacco rattle virus, a potato virus X, a pea early browning virus, or a barley stripe mosaic virus.

11. The method of claim 1, wherein the plant is monocotyledonous.

12. The method of claim 11, wherein the plant is wheat, maize, or Setaria.

13. The method of claim 1, wherein the plant is dicotyledonous.

14. The method of claim 13, wherein the plant is tomato, soybean, tobacco, potato, or Arabidopsis.