ENGINEERING PLANT GENOMES USING CRISPR/Cas SYSTEMS

• US 20150167000A1
• Filed: 02/24/2015
• Published: 06/18/2015
• Est. Priority Date: 03/15/2013
• 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., Atkins, Paul, Baltes, Nicholas J.
• Application Number:
  US14/629,859
• Publication Number:
  US 20150167000A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8203 Virus mediated transformation
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 2750/00043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/21 Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)

Abstract

Materials and methods for gene targeting using Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) systems are provided herein.

Claims

1. A method for modifying the genomic material in a plant cell, comprising:

(a) introducing into the plant cell a nucleic acid molecule, wherein the nucleic acid molecule comprises a crRNA and a tracrRNA, a chimeric cr/tracrRNA hybrid, a sequence encoding the crRNA and the tracrRNA, or a sequence encoding the chimeric cr/tracrRNA hybrid, wherein the crRNA and tracrRNA, or the chimeric cr/tracrRNA hybrid, is targeted to a sequence that is endogenous to the plant cell; and

(b) introducing into the plant cell a Cas9 endonuclease molecule or a nucleic acid molecule comprising a sequence encoding the Cas9 endonuclease molecule, wherein the Cas9 endonuclease molecule induces a double strand break at or near the sequence to which the crRNA and tracrRNA sequence is targeted, or at or near the sequence to which the chimeric cr/tracrRNA hybrid is targeted.

2. The method of claim 1, wherein step (a) comprises delivering to the plant cell the nucleic acid molecule encoding the crRNA and tracrRNA or the chimeric cr/tracrRNA hybrid, and step (b) comprises delivering to the plant cell the nucleic acid molecule comprising the sequence encoding the Cas9 endonuclease molecule.

3. The method of claim 2, wherein the delivering in step (a), step (b), or both steps (a) and (b) comprises delivery via a DNA virus.

4. The method of claim 3, wherein the DNA virus is a geminivirus.

5. The method of claim 2, wherein the delivering in step (a), step (b), or both steps (a) and (b) comprises delivery via RNA virus.

6. The method of claim 5, wherein the RNA virus is a tobravirus.

7. The method of claim 2, wherein the delivering in step (a), step (b), or both steps (a) and (b) comprises delivery to protoplasts.

8. The method of claim 2, wherein the delivering in step (a), step (b), or both steps (a) and (b) comprises T-DNA delivery.

9. The method of claim 8, wherein the T-DNA delivery is via Agrobacterium or Ensifer.

10. The method of claim 2, wherein the delivering in step (a), step (b), or both steps (a) and (b) comprises particle bombardment.

11. The method of claim 2, wherein the sequence encoding the Cas9 endonuclease molecule is operably linked to a promoter that is constitutive, cell specific, inducible, or activated by alternative splicing of a suicide exon.

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

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

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

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