METHODS AND COMPOSITIONS FOR NUCLEASE-MEDIATED TARGETED INTEGRATION OF TRANSGENES

• US 20130326645A1
• Filed: 05/07/2013
• Published: 12/05/2013
• 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 Biosciences, Inc.
• Inventors: Urnov, Fyodor, Sastry-Dent, Lakshmi, Cost, Gregory J., Ainley, William M., Petolino, Joseph F., Samuel, Jayakumar Pon, Webb, Steven R.
• Granted Patent: US 10,174,331 B2
• Time in Patent Office: Days
• US Class Current: 800/14
• 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 double-stranded polynucleotide comprising:

an exogenous nucleic acid sequence;

a sequence comprising one or more target sites for one or more nucleases,

wherein the one or more target sites are not within the exogenous nucleic acid sequence and further wherein the double-stranded polynucleotide does not comprise homology arms.

2. The double-stranded polynucleotide of claim 1, wherein the exogenous nucleic acid sequence is at least 1 kb in length.

3. The double-stranded polynucleotide of claim 1, wherein the exogenous nucleic acid sequence polynucleotide is a plasmid.

4. The double-stranded polynucleotide of claim 1, wherein the exogenous nucleic acid sequence comprises a transgene.

5. The double-stranded polynucleotide of claim 1, wherein the sequence comprises two target sites and a spacer of at least 5 nucleotides between the two target sites.

6. A cell comprising the double-stranded polynucleotide of claim 1.

7. A method of integrating an exogenous nucleic acid sequence polynucleotide into an endogenous locus of a cell, the method comprising;

introducing a double-stranded polynucleotide according to claim 1 into the cell;

introducing one or more nucleases into the cell, wherein the nucleases cleave the double-stranded polynucleotide and cleave the endogenous locus such that the exogenous nucleic acid sequence polynucleotide is integrated into the endogenous locus.

8. The method of claim 7, wherein the exogenous nucleic acid sequence polynucleotide is integrated in a forward orientation.

9. The method of claim 7, wherein the exogenous nucleic acid sequence polynucleotide is integrated in a reverse orientation.

10. The method of claim 7, wherein the same nucleases cleave the endogenous locus and the donor polynucleotide.

11. The method of claim 7, wherein different nucleases cleave the endogenous locus and the donor polynucleotide.

12. The method of claim 7, wherein the exogenous nucleic acid sequence polynucleotide is integrated into the endogenous locus via homology-independent mechanisms.

13. The method of claim 7, wherein the nucleic acid comprising the two target sites and the spacer is non-naturally occurring such that the target sites are not re-created following integration of the exogenous nucleic acid sequence polynucleotide.

14. The method of claim 7, wherein the nucleases generate a deletion in the endogenous locus and the exogenous nucleic acid sequence polynucleotide is integrated into the deletion.

15. The method of claim 7, wherein the cell is a eukaryotic cell.

16. The method of claim 15, wherein the cell is plant or mammalian cell.

17. The method of claim 16, wherein the plant cell is a dicotyledonous or a monocotyledonous plant cell.

18. A transgenic organism comprising an exogenous nucleic acid sequence polynucleotide integrated according to the method of claim 7.