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 dicot genomic DNA, comprising:
a) obtaining a first dicot plant comprising a mutation in a centromeric histone 3 (CENH3) gene and optionally a maize CENH3 tailswap transgene, wherein said first dicot plant expresses a DNA modification enzyme and optionally at least one guide nucleic acid;
b) obtaining a second dicot plant, wherein the second dicot plant comprises the dicot plant genomic DNA which is to be edited;
c) pollinating the first dicot plant with pollen from the second dicot 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 dicot plant but not the first dicot 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 dicot plant;
wherein the mutation in a CENH3 gene is selected from the group consisting of a loss-of-function mutation, a partial loss-of-function mutation, a restored frameshift mutation, and an in-frame deletion mutation.
2. The method of claim 1 further comprising:
e) allowing the at least one haploid progeny selected in step (d) to undergo chromosome doubling.
3. The method of claim 2 , wherein the edited haploid progeny undergoes spontaneous chromosome doubling, thereby creating an edited doubled haploid progeny.
4. The method of claim 2 , 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 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 (Cas12a), 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, dCpf1 non-FokI nuclease, chimeric Cpf1-cytidine deaminase, and Cpf1-adenine deaminase.
7. The method of claim 1 , wherein the at least one guide nucleic acid is a guide RNA.
8. The method of claim 7 , wherein the guide RNA is an 18-21 nucleotide sequence and is at least 90% identical to a target sequence.
9. The method of claim 8 , wherein the target sequence is SEQ ID NO: 103, and wherein the dicot genomic DNA is Arabidopsis genomic DNA.
10. The method of claim 1 , wherein the first dicot plant expresses a marker gene.
11. The method of claim 10 , wherein the marker gene is selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, B1, C1, R-nj, anthocyanin pigments.
12. The method of claim 1 , wherein the first dicot plant is a transformable dicot plant selected and/or derived from the group consisting of Arabidopsis, Brassica , soybean, tomato, sunflower, and cotton.
13. The method of claim 1 , wherein the partial loss-of-function mutation is a frame-shift mutation near the 3′ terminus of the CENH3 gene.
14. The method of claim 1 , wherein the loss-of-function mutation is a knock-out mutation.
15. The method of claim 1 , wherein the at least one haploid progeny comprises SEQ ID NO: 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, or 117, and further wherein the at least one haploid progeny is Arabidopsis.