Targeted integration into the Zp15 locus

• US 8,329,986 B2
• Filed: 12/17/2009
• Issued: 12/11/2012
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Butler, Holly J., Corbin, David R., Doyon, Yannick, Gao, Zhifang, Shukla, Vipula K., Urnov, Fyodor, Worden, Sarah E.
• Primary Examiner(s): O Hara, Eileen B
• Application Number:
  US12/653,735
• Publication Number:
  US 20100199389A1
• Time in Patent Office: 1,090 Days
• Field of Search: None
• US Class Current: 800/278
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into a plant Zp15 locus, for example, for expression of a polypeptide of interest.

Claims

1. A method of integrating one or more exogenous nucleic acid sequences into the genome of a plant cell, the method comprising:

making a double-stranded cleavage in the genome of the plant cell in a Zp15 locus using a zinc finger nuclease comprising a zinc finger binding domain that binds to a target site selected from the group shown in Table 1, thereby resulting in integration of a polynucleotide comprising the one or more exogenous sequences into the genome of the cell in the Zp15 locus.

2. The method of claim 1, further comprising expressing a product of the one or more exogenous sequences.

3. The method of claim 1, wherein the double-stranded cleavage is made by

(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site selected from the group shown in Table 1 in a Zp15 locus in the genome of the plant cell;

and

(b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the Zp15 locus in the genome of the plant cell, wherein the second target site is different from the first target site; and

wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the plant cell in the Zp15 locus is cleaved.

4. The method of claim 3, wherein the zinc finger binding domains are selected from the group shown in Table 1.

5. The method of claim 3, wherein the cleavage half-domains are naturally or non-naturally occurring.

6. The method of claim 1, wherein the one or more exogenous nucleic acid sequences comprise a coding sequence, a regulatory sequence, or a target site for a DNA-binding domain.

7. The method of claim 6, wherein the coding sequence encodes for a product that confers:

herbicide resistance;

herbicide tolerance;

insect resistance;

insect tolerance;

disease resistance;

disease tolerance;

stress tolerance;

stress resistance;

a change in oxidative stress;

increased yields of oil;

a change in food content and makeup;

a change in physical appearance;

male sterility;

drydown;

standability;

prolificacy;

a change in starch quantity or quality;

a change in oil quality;

a change in protein quality or quantity;

a change in amino acid composition or combinations thereof.

8. The method of claim 1, wherein the polynucleotide further comprises nucleotide sequences that are homologous to sequences in the Zp15 locus.

9. The method according to claim 8, wherein the homologous nucleotide sequences flank the exogenous sequence.

10. The method of claim 1, wherein the polynucleotide further comprises a promoter.

11. The method of claim 1, wherein one or more of the integrated exogenous sequences are transmitted to progeny in subsequent generations.

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

13. The method of claim 12, wherein the plant cell is a maize cell.

14. A plant or plant part, comprising one or more exogenous sequences integrated into the Zp15 locus by the method of claim 1.

15. A seed comprising one or more exogenous sequences integrated into the Zp15 locus by the method of claim 1.