Methods and compositions for nuclease-mediated targeted integration of transgenes

• US 10,174,331 B2
• Filed: 05/07/2013
• Issued: 01/08/2019
• Est. Priority Date: 05/07/2012
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Inventors: Cost, Gregory J., Urnov, Fyodor, Ainley, W. Michael, Petolino, Joseph F., Samuel, Jayakumar Pon, Webb, Steven R., Sastry-Dent, Lakshmi
• Primary Examiner(s): Keogh, Matthew R
• Application Number:
  US13/889,162
• Publication Number:
  US 20130326645A1
• Time in Patent Office: 2,072 Days
• Field of Search: None
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8278 Sulfonylurea
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

Disclosed herein are methods and compositions for homology-independent targeted insertion of donor molecules into the genome of a cell.

Claims

1. A method of integrating a transgene into an endogenous locus of a cell, the method comprising;

introducing one or more nucleases into the cell, the cell comprising a circular double-stranded plasmid donor, the double-stranded plasmid donor comprising;

a transgene; and

first and second sequences flanking the transgene, the first and second sequences flanking the transgene comprising paired target sites for one or more nucleases, the target sites flanking a spacer sequence, wherein at least one spacer sequence differs from the spacer sequence present in the genome of the cell, wherein the double-stranded plasmid does not comprise homology arms and further wherein the nucleases cleave the double-stranded plasmid in the sequences flanking the transgene and cleave the endogenous locus such that the transgene is integrated into the endogenous locus via homology-independent mechanisms.

2. The method of claim 1, wherein the transgene is integrated in a forward orientation.

3. The method of claim 1, wherein the transgene is integrated in a reverse orientation.

4. The method of claim 1, wherein the same nucleases cleave the endogenous locus and the double-stranded plasmid.

5. The method of claim 1, wherein different nucleases cleave the endogenous locus and the double-stranded plasmid.

6. The method of claim 1, wherein the spacer sequences comprise at least 5 nucleotides.

7. The method of claim 1, wherein the nucleases generate a deletion in the endogenous locus and the transgene is integrated into the deletion.

8. The method of claim 1, wherein the cell is a eukaryotic cell.

9. The method of claim 8, wherein the cell is a plant or mammalian cell.

10. The method of claim 9, wherein the plant cell is a dicotyledonous or a monocotyledonous plant cell.