US 12,703,869 B2Granted

Intra-genomic homologous recombination

AssigneePioneer Hi-Bred International, Inc.

InventorsAjith Anand, Maren L Arling, Pierluigi Barone, Hyeon-Je Cho, William James Gordon-Kamm, Sandeep Kumar, Brian Lenderts, Keith S Lowe, Sergei Svitashev, Xinli Emily Wu

Life of this patent

Estimating the term…

patentgrep

Loading patent…

Abstract

Compositions and methods are provided for an inducible, high efficiency intra-genomic homologous recombination (IGHR) system in plants, that can be used for non-chimeric, heritable gene targeting. The advantage of IGHR approach to targeted integration is that every cell contains donor DNA and nuclease-encoding sequences, so that there are many potentially homology-directed targeting events that can occur during plant development.

Claims18

Dependent claims

A method for intra-genomic homologous recombination at a target site in a plant cell in a single generation, comprising:

(a) providing to the plant cell a composition comprising a construct, wherein the construct comprises:
(i) a donor DNA flanked by homology regions that are capable of hybridizing with a sequence near the target site, wherein the donor DNA molecule flanked by homology regions is further flanked by Cas endonuclease cleavage site sequences, wherein the Cas endonuclease cleavage site sequences share homology with a sequence at the target site, wherein the donor DNA, homology regions, and Cas endonuclease cleavage sites are positioned in the construct between a promoter and a selectable marker coding sequence, thereby rendering the selectable marker non-functional;
(ii) a gene encoding a Cas endonuclease, operably linked to an inducible promoter expressed to result in delayed induction of the Cas endonuclease;
and
(iii) at least one gene cassette comprising at least one morphogenic factor;
wherein the construct is integrated into an integration site in the genome of the plant cell that is not the target site;
(b) providing to the plant cell a guide RNA, wherein the guide RNA recognizes a sequence at or near the target site in the plant cell;
(c) culturing the plant cell to an undifferentiated stage via an extended tissue culture;
(d) incubating the plant cell under a condition that induces the inducible promoter of (a) (ii), such that the gene encoding the Cas endonuclease of (a) (ii) is expressed;
(e) allowing the Cas endonuclease of (a) (ii) to form a complex with the guide RNA of (b);
(f) cleaving the target site and the donor DNA flanked by homology regions with the Cas endonuclease;
(g) releasing the donor DNA from the integration site, thereby activating the expression of the selectable marker;
(h) incorporating the donor DNA into the target site;
(i) incubating the plant cell under a condition that results in excision of the at least one morphogenic factor;
(j) releasing the at least one morphogenic factor;
(k) verifying release of the donor DNA from the integration site by evaluating the expression of the activated selectable marker;
and
(l) verifying the incorporation of the donor DNA into the target site;
wherein the donor DNA is heterologous to the target site.

16 more elements

A method of producing a plant with a stably integrated donor DNA in a single generation, the method comprising:

(a) providing to a cell obtained or derived from the plant a composition comprising a construct, wherein the construct comprises:
(i) a donor DNA flanked by homology regions that are capable of hybridizing with a sequence near the target site, wherein the donor DNA molecule flanked by homology regions is further flanked by Cas endonuclease cleavage site sequences, wherein the Cas endonuclease cleavage site sequences share homology with a sequence at the target site, wherein the donor DNA, homology regions, and Cas endonuclease cleavage sites are positioned in the construct between a promoter and a selectable marker coding sequence, thereby rendering the selectable marker non-functional;
(ii) a gene encoding a Cas endonuclease, operably linked to an inducible promoter expressed to result in delayed induction of the Cas endonuclease;
and
(iii) at least one gene cassette comprising at least one morphogenic factor;
wherein the construct is integrated into an integration site in the genome of the cell that is not the target site;
(b) providing to the cell a guide RNA, wherein the guide RNA recognizes a sequence at or near the target site in the cell;
(c) culturing the cell to an undifferentiated stage via an extended tissue culture;
(d) incubating the cell under a condition that induces the inducible promoter of (a) (ii), such that the gene encoding the Cas endonuclease of (a) (ii) is expressed;
(e) allowing the Cas endonuclease of (a) (ii) to form a complex with the guide RNA of (b);
(f) cleaving the target site and the donor DNA flanked by homology regions with the Cas endonuclease;
(g) releasing the donor DNA from the integration site, thereby activating the expression of the selectable marker;
(h) incorporating the donor DNA into the target site;
(i) incubating the cell under a condition that results in excision of the at least one morphogenic factor;
(j) releasing the at least one morphogenic factor;
(k) selecting one or more cells by evaluating the expression of the selectable marker;
(l) obtaining or deriving a plant from the one or more cells of (k);
wherein the donor DNA is heterologous to the target site.

15 more elements

Dependencies are inferred from the claim text.

Facts

Application: 17/904,722Filed 22 Feb 2021 · us provisional application
Priority: 24 Feb 2020
Published: US 2023/0091338 A123 Mar 2023
Granted: 11 Aug 2026
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

C12N 15/8213
Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for…:…
A01H 6/4684
Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize: zea mays [maize]
A01H 6/542
Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut: glycine max [soybean]
C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]