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 maize genomic DNA, comprising:
a) obtaining a first maize plant comprising a loss-of-function mutation in a wildtype patatin-like phospholipase A2α gene having a cDNA sequence as represented by SEQ ID NO: 19, wherein said first maize plant expresses a DNA modification enzyme and at least one guide nucleic acid;
b) obtaining a second maize plant, wherein the second maize plant comprises the plant genomic DNA which is to be edited;
c) pollinating the second maize plant with pollen from the first maize 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 maize plant but not the first maize plant, and the genome of the haploid progeny has been modified by the DNA modification enzyme and at least one guide nucleic acid delivered by the first maize plant;
wherein the DNA modification enzyme is a base editor.
2. The method of claim 1 , wherein the DNA modification enzyme comprises a site-directed nuclease selected from the group consisting of a Cas9 nuclease, Cpf1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, 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 optional 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.
7. The method of claim 1 , wherein the first plant expresses a marker gene.
8. The method of claim 7 , wherein the marker gene is selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, B1, C1, R-nj, and anthocyanin pigments.
9. The method of claim 1 , wherein the first plant is a transformable maize plant selected and/or derived from the group consisting of Stock 6, RWK, RWS, UH400, AX5707RS, and NP2222-matl.
10. The method of claim 1 , wherein the base editor comprises a cytidine deaminase fused to a Cas polypeptide.
11. The method of claim 10 , wherein the cytidine deaminase is an APOBEC deaminase.
12. The method of claim 1 , wherein the base editor comprises an adenine deaminase fused to a Cas polypeptide.
13. The method of claim 1 , wherein the base editor comprises a uracil DNA glycosylase fused to a Cas polypeptide.