SIMULTANEOUS GENE EDITING AND HAPLOID INDUCTION
The presently disclosed subject matter relates to using a haploid inducing line (whether existing or created) and transforming the haploid line so that it encodes cellular machinery capable of editing genes. The transformed haploid inducing line is used as a parent in a cross between two plants. During pollination, the parental gametes fuse to form an embryo; and the gene editing machinery is also delivered to the embryo at this time. During embryonic development, one set of parental chromosomes are lost, and the gene editing machinery operates on the remaining set of chromosomes. Thus, at least one haploid progeny with edited genes is produced from the cross.
1 . A method of editing monocot plant genomic DNA, comprising:
a) obtaining a first monocot plant comprising a mutation in a patatin-like phospholipase gene, and wherein said first monocot plant is capable of expressing a DNA modification enzyme and optionally at least one guide nucleic acid;
b) obtaining a second monocot plant, wherein the second monocot plant comprises the monocot plant genomic DNA which is to be edited;
c) pollinating the second monocot plant with pollen from the first monocot plant; and
d) selecting at least one haploid progeny produced by the pollination of step (c) wherein the haploid progeny comprises the genome of the second monocot plant but not the first monocot plant, and the genome of the haploid progeny has been modified by the DNA modification enzyme and optional at least one guide nucleic acid delivered by the first monocot plant.
2 . The method of claim 1 , wherein the DNA modification enzyme is a site-directed nuclease selected from the group consisting of meganucleases (MNs), zinc-finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), Cas9 nuclease, Cpf1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FEN1-FokI, and Mega-TALs, a nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease and dCpf1 non-FokI nuclease.
3 . The method of claim 1 , wherein the at least one guide nucleic acid is a guide RNA.
4 . The method of claim 1 , wherein the edited haploid progeny is treated with a chromosome doubling agent, thereby creating an edited doubled haploid progeny.
5 . The method of claim 4 , wherein the chromosome doubling agent is colchicine, pronamide, dithipyr, trifluralin, or another known anti-microtubule agent.
6 . The method of claim 1 , wherein the first monocot plant is selected from the group consisting of maize, wheat, rice, barley, oats, triticale, sorghum, pearl millet, teosinte, bamboo, sugar cane, asparagus, onion, and garlic.
7 . The method of claim 1 , wherein the second monocot plant is selected from the group consisting of maize, wheat, rice, barley, oats, triticale, sorghum, pearl millet, teosinte, bamboo, sugar cane, asparagus, onion, and garlic.
8 . The method of claim 3 , wherein the guide RNA is an 18-21 nucleotide sequence and is homologous to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 8, 21, 23, 25, 29, 32, and 33.
9 . The method of claim 1 , wherein the first monocot plant expresses a marker gene.
10 . The method of claim 9 , wherein the marker gene is selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, B1, C1, R-nj, and anthocyanin pigments.
11 . A gene-edited monocot plant produced by the method of claim 1 .