	US 12,331,329 B2
	Genome editing in plants
Yurong Chen, Chesterfield, MO (US); Annie Saltarikos, O'Fallon, MO (US); Jianping Xu, Chesterfield, MO (US); and Xudong Ye, Chesterfield, MO (US)
Assigned to Monsanto Technology LLC, St. Louis, MO (US)
Appl. No. 17/045,747
Filed by Monsanto Technology LLC, St. Louis, MO (US)
PCT Filed May 24, 2019, PCT No. PCT/US2019/033984 § 371(c)(1), (2) Date Oct. 6, 2020, PCT Pub. No. WO2019/227030, PCT Pub. Date Nov. 28, 2019.
Claims priority of provisional application 62/676,228, filed on May 24, 2018.
Prior Publication US 2021/0054390 A1, Feb. 25, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/82 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01)

CPC C12N 9/22 (2013.01) [C12N 15/8201 (2013.01); C12N 15/8213 (2013.01); C12N 15/113 (2013.01); C12N 15/8205 (2013.01); C12N 2310/20 (2017.05)]

25 Claims

1. A method of editing a genome of a plant, comprising:

a) excising an embryo from a dry mature plant seed, thereby producing a dry-excised mature plant embryo explant;

b) delivering to the dry-excised mature plant embryo explant via particle bombardment a recombinant DNA construct comprising a sequence encoding a site-specific nuclease, wherein

i) the site-specific nuclease is a ribonucleoprotein;

ii) the site-specific nuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csn1, Csx12, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, an Argonaute protein, or a homolog or modified version thereof;

iii) the site-specific nuclease is not a guided nuclease; or

iv) the site-specific nuclease is a meganuclease, a zinc-finger nuclease (ZFN), a recombinase, a transposase, or a transcription activator-like effector nuclease (TALEN), and

wherein the sequence encoding the site-specific nuclease is operably linked to a plant-expressible promoter,

c) culturing the dry-excised mature plant embryo explant after bombardment for at least two days in dark at about 37° C.; and

d) regenerating a plant from the dry-excised mature plant embryo explant without forming an embryogenic callus or callus culture from the dry-excised mature plant embryo explant,

wherein the regenerated plant comprises an edit or site-directed integration at or near the target site of the site-specific nuclease in the genome of at least one cell of the regenerated plant.