Targeted genomic alteration

• US 20110189775A1
• Filed: 01/24/2011
• Published: 08/04/2011
• Est. Priority Date: 01/22/2010
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Zeitler, Bryan, Murray, Michael G., Urnov, Fyodor, Ainley, William M.
• Granted Patent: US 10,260,062 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A01H 1/06 Processes for producing mutations, e.g. treatment with chemicals or with radiation specific mutations prepared by genetic engineering on plant cell or plant tissues C12N15/00 ; process for producing transgenic plants C12N15/82
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for targeted integration and/or targeted excision of one or more sequences into a cell, for example, for expression of one or more polypeptides of interest.

Claims

1. An isolated nucleic acid molecule comprising a plurality of paired target sites for one or more pairs of zinc finger nucleases.

2. The nucleic acid molecule of claim 1, where one target site from each paired target site comprises the same sequence.

3. The nucleic acid molecule of claim 1, further comprising one or more coding sequences.

4. A cell or cell line comprising a nucleic acid molecule according to claim 1.

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

6. The cell of claim 5, wherein the eukaryotic cell is a plant or mammalian cell.

7. The cell of claim 4, wherein the nucleic acid molecule is integrated into the genome of the cell.

8. A method for integrating one or more exogenous sequences into the genome of a cell, the method comprising

(a) providing one or more pairs of zinc finger nucleases to a cell according to claim 7, wherein the zinc finger nucleases bind to a target site in the integrated nucleic acid molecule, such that binding of the nucleases to their target sites cleaves the genome of the cell;

and

(b) contacting the cell with a polynucleotide comprising an exogenous sequence, wherein the exogenous sequence is integrated into the genome of cell within the integrated nucleic acid.

9. The method of claim 8, further comprising repeating steps (a) and (b) with additional zinc finger nucleases that cleave additional target sites in the integrated nucleic acid molecule in the presence of additional exogenous sequences, thereby inserting the additional exogenous sequences into the genome of the cell.

10. The method of claim 8, wherein one or more of the exogenous sequences comprise one or more target sites for zinc finger nucleases.

11. The method of claim 8, wherein the target site is a zinc finger nuclease half target site, wherein, upon integration of the half target site, a target site is created.

12. The method of claim 8, wherein one or more of the exogenous sequences comprises a coding sequence and the cell expresses the product of the coding sequence.

13. A method of deleting one or more sequences inserted into the genome of a cell, the method comprising,

(a) integrating a plurality of exogenous sequences according to claim 9;

and

(b) expressing the appropriate nucleases in the cell such that one or more of the exogenous sequences are deleted from the genome.

14. A method of providing a genomically altered cell, the method comprising

(a) integrating one or more exogenous sequences in at least a first cell according to the method of claim 8;

(b) allowing the first cell to develop into a sexually mature organism; and

(c) crossing the organism with a second organism comprising at least one genomic alteration to generate a second cell with with at least one of the genomic alterations of the first and second organisms.

15. The method of claim 14, wherein the second cell genomic alterations comprise a plurality of heterologous genes at a single genomic location in the second cell.

16. The method of claim 14, wherein the first cell further comprises on one more deletions within the integrated nucleic acid molecule.

17. The method of claim 8, wherein the cell further comprises modifications of its genome outside the integrated nucleic acid molecule.

18. The method of claim 8, wherein the cell is a plant cell.