Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes

• US 8,889,390 B2
• Filed: 02/13/2013
• Issued: 11/18/2014
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc.
• Inventors: Gupta, Manju, Palta, Asha M., Novak, Stephen, Urnov, Fyodor, Gopalan, Sunita
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US13/766,380
• Publication Number:
  US 20130145503A1
• Time in Patent Office: 643 Days
• Field of Search: 435/183
• US Class Current: 435/183
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 9/1092 3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase

Abstract

The present disclosure relates to engineered zinc finger proteins that target 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) genes in plants and methods of using such zinc finger proteins in modulating gene expression, gene inactivation, and targeted gene modification. In particular, the disclosure pertains to zinc finger nucleases for targeted cleavage and alteration of EPSPS genes.

Claims

1. A donor vector comprising nucleic acid sequences having homology to chromosomal 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) DNA sequences, said nucleic acid sequences comprising a first, second and a third DNA sequences;

wherein the first and second sequences comprise a sequence as shown in any one of SEQ ID NOs;

141, 142, and 144-153; and

the third sequence is interposed between the first and second sequences and comprises an exogenous sequence.

2. The donor vector of claim 1, wherein the third sequence comprises sequences encoding a selectable marker.

3. The donor vector of claim 2, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β

-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, hygromycin phosphotransferase, β

-lactamase, catechol dioxygenase, α

-amylase, tyrosinase, β

-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.

4. The donor vector of claim 1, wherein the third sequence comprises sequences selected from the group consisting of a sequence encoding a protein other than a selectable marker;

one or more transcriptional regulatory sequences;

one or more sequences which enhance or diminish protein targeting;

one or more sequences encoding a portion of a protein;

a small interfering RNA; and

a micro RNA.

5. The donor vector of claim 4, wherein the third sequence comprises a sequence encoding a mutant EPSPS chromosomal sequence that increases tolerance of a plant to the herbicide glyphosate.