Site specific integration of a transgne using intra-genomic recombination via a non-homologous end joining repair pathway

Patent metadata

Field	Value
Publication number	US-2018142249-A1
Application number	US-201715797285-A
Country	US
Kind code	A1
Filing date	Oct 30, 2017
Priority date	Nov 21, 2016
Publication date	May 24, 2018
Grant date	—

Abstract

Compositions and methods to modify at least one target locus in a plant cell are provided, which comprises providing a plant cell, a plant, or a plant part with one or more target loci and one or more donor loci, providing at least one cleaving site specific nuclease to produce a double strand break within the target loci, followed by non-homologous end joining of at least one donor locus within at least one target locus. Target loci, donor loci and nuclease loci used in these methods, and plant cells, plants and plant parts comprising these target loci, donor loci, nuclease loci and/or the recombined loci are also provided.

First claim

What is claimed is:

1 . A method for inserting an integrated donor DNA within a plant genomic target locus, the method comprising:
a) providing a first viable plant containing a genomic DNA, the genomic DNA comprising the donor DNA flanked by a plurality of recognition sequences and the plant genomic target locus, wherein the plant genomic target locus comprises at least one recognition sequence; b) providing a second viable plant containing a genomic DNA, the genomic DNA comprising a DNA encoding at least one zinc finger nuclease engineered to cleave the genomic DNA at the recognition sequence; c) crossing the first and second viable plants such that F1 seed is produced on either the first or the second viable plant; d) expressing the zinc finger nuclease within the F1 seed or a F1 plant, wherein the expressed zinc finger nuclease cleaves the donor DNA and the genomic DNA at the recognition sequence; and e) growing the resultant F1 plant containing a genomic DNA, wherein the donor DNA is integrated within the recognition sequence of the plant genomic target locus via non-homologous end joining.

2 . The method of claim 1 , wherein the recognition sequence comprises a first and second recognition sequence.

3 . The method of claim 2 , wherein the first and second recognition sequences are identical.

4 . The method of claim 3 , wherein the zinc finger nuclease is provided by crossing the first and second viable plants such that the zinc finger nuclease cleaves both recognition sequences.

5 . The method of claim 1 , wherein the donor DNA and the plant genomic target locus are unlinked.

6 . The method of claim 5 , wherein the donor DNA and the plant genomic target locus are located on homologous chromosomes, or on non-homologous chromosomes.

7 . The method of claim 1 , wherein the plant genomic target locus of step a) further comprises an expression cassette located:
a) between the first and second recognition sequences; or b) outside of the first recognition sequence; or c) outside of the second recognition sequence.

8 . The method of claim 1 , wherein the first viable plant is homozygous for at least one genomic target locus or is homozygous for at least one donor DNA.

9 . The method of claim 1 , wherein the first viable plant is heterozygous for at least one genomic target locus or is heterozygous for at least one donor DNA.

10 . The method of claim 1 , wherein the plant genomic target locus is:
a) a transgenic locus; or b) an endogenous locus.

11 . The method of claim 1 , wherein the zinc finger nuclease is driven by a promoter selected from the group consisting of a pollen-specific promoter, a seed-specific promoter, and a developmental-stage specific promoter.

12 . The method of claim 1 , wherein the donor DNA comprises a selectable marker.

13 . A method for transmitting a transgene into other plants, the method comprising:
a) crossing a first plant regenerated from a plant cell or tissue transformed with an isolated nucleic acid molecule comprising a genomic target locus and the transgene with a second plant regenerated from a plant cell or tissue transformed with an isolated nucleic acid molecule comprising a promoter operably linked to a zinc finger nuclease; b) expressing the zinc finger nuclease so that a first zinc finger nuclease monomer is paired with a second zinc finger nuclease monomer; c) obtaining a F1 plant resulting from the cross wherein the transgene is specifically and stably integrated within the genomic target locus via non-homologous end joining; and d) cultivating the F1 plant resulting from the cross.

14 . The method of claim 13 , wherein the plant regenerated from the plant cell or tissue transformed with the isolated nucleic acid molecule comprising the promoter operably linked to the zinc finger nuclease comprises at least one zinc finger nuclease monomer.

15 . The method of claim 14 , wherein the plant regenerated from the plant cell or tissue transformed with the isolated nucleic acid molecule comprising the promoter operably linked to the zinc finger nuclease comprises the first and the second zinc finger nuclease monomer.

16 . The method of claim 13 , wherein the plant regenerated from the plant cell or tissue transformed with the isolated nucleic acid molecule comprising the promoter operably linked to the zinc finger nuclease comprises the first zinc finger nuclease monomer.

17 . The method of claim 16 , wherein the plant regenerated from the plant cell or tissue transformed with the isolated nucleic acid molecule comprising the genomic target locus and the transgene further comprises an isolated nucleic acid molecule comprising a promoter operably linked to a second zinc finger nuclease, wherein the second zinc finger nuclease comprises the second zinc finger nuclease monomer.

18 . The method of claim 13 , wherein the pairing of the first and second zinc finger nuclease monomers of step b) results in the release of the transgene and cleavage of the genomic target locus.

19 . The F1 plant according to claim 1 or 13 , further comprising a transgenic event.

20 . The F1 plant of claim 19 , wherein the transgenic event comprises an agronomic trait.

21 . The F1 plant of claim 20 , wherein the agronomic trait is selected from the group consisting of an insecticidal resistance trait, herbicide tolerance trait, nitrogen use efficiency trait, water use efficiency trait, nutritional quality trait, DNA binding trait, small RNA trait, selectable marker trait, or any combination thereof.

22 . The F1 plant of claim 20 , wherein the agronomic trait comprises a herbicide tolerant trait.

23 . The F1 plant of claim 22 , wherein the herbicide tolerant trait comprises a dgt-28 coding sequence.

24 . The F1 plant of claim 21 , wherein the transgenic plant produces a commodity product.

25 . The F1 plant of claim 24 , wherein the commodity product is selected from the group consisting of protein concentrate, protein isolate, grain, meal, flour, oil, or fiber.

26 . The F1 plant of claim 25 , wherein the transgenic plant is selected from the group consisting of a dicotyledonous plant or a monocotyledonous plant.

27 . The F1 plant of claim 26 , wherein the monocotyledonous plant is a Zea mays plant.

28 . The F1 plant of claim 26 , wherein the dicotyledonous plant is a tobacco plant.

Assignees

• Dow Agrosciences Llc

Inventors

• Kumar Sandeep
• Petolino Joseph F
• Worden Andrew F
• Barone Pierluigi
• Simpson Matthew A
• Strange Tonya L

Classifications

• C12N15/8213 Primary

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C12N15/8234

  Seed-specific, e.g. embryo, endosperm · CPC title

• C12N15/8286

  for insect resistance · CPC title

• C12N15/8261

  with agronomic (input) traits, e.g. crop yield · CPC title

• C12N15/8209

  Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers · CPC title