Methods and compositions for genome editing via haploid induction
Methods and compositions for improved plant breeding using gene editing and haploid induction are provided.
1. A method of editing maize genomic DNA, comprising:
a) introducing into the genome of a maize haploid inducer line a nucleic acid sequence encoding at least one Genome Editing Component (GEC) to produce a first maize plant, wherein the genome of the maize haploid inducer comprises a supernumerary chromosome, and wherein the at least one GEC is introduced into the supernumerary chromosome;
b) providing a second maize plant, wherein the second maize plant comprises the maize plant genomic DNA which is to be edited;
c) crossing the second maize plant with the first maize plant; and
d) selecting at least one haploid progeny produced by the cross of step (c) wherein the haploid progeny comprises the genome of the second maize plant and the supernumerary chromosome from said first maize plant but otherwise lacks the genome of the first maize plant, and wherein the genome of the haploid progeny comprises at least one targeted genome modification.
2. The method of claim 1 , wherein the at least one GEC comprises at least one recombinase or at least one endonuclease.
3. The method of claim 2 , wherein the at least one endonuclease is selected from the group consisting of a CRISPR associated nuclease, a transcription activator-like effector nuclease (TALEN), a TALE-like protein, a zinc finger nuclease, and a meganuclease.
4. The method of claim 2 , wherein the at least one recombinase is selected from the group consisting of a tyrosine recombinase attached to a DNA recognition motif and a serine recombinase attached to a DNA recognition motif.
5. The method of claim 4 , wherein the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Gin recombinase, a FLP recombinase, and a Tnp1 recombinase.
6. The method of claim 4 , wherein the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a Bxb1 integrase, a phiC31 integrase, an R4 integrase, and a TP-901 integrase.
7. The method of claim 1 , wherein the edited haploid progeny is treated with a chromosome doubling agent, thereby creating an edited doubled haploid progeny.
8. The method of claim 7 , wherein the chromosome doubling agent is colchicine, pronamide, and trifluralin.
9. The method of claim 1 , wherein the first maize plant expresses a marker gene.
10. The method of claim 9 , wherein the marker gene is selected from the group consisting of GFP and anthocyanin pigments.
11. The method of claim 1 , wherein the maize haploid inducer line comprises a knock-out mutation in a patatin-like phospholipase gene.
12. A gene-edited-haploid progeny produced by the method of claim 1 , wherein the gene-edited haploid progeny comprises at least one targeted genome modification, and wherein the gene-edited haploid progeny comprises the genome of the second maize plant and the supernumerary chromosome from said first maize plant but otherwise lacks the genome of the first maize plant.
13. The method of claim 1 , wherein the haploid inducer line further comprises a high-oil or redroot trait.
14. The method of claim 2 , wherein the endonuclease is Cpf1.
15. A method of editing maize genomic DNA, comprising:
a) introducing into the genome of a maize haploid inducer line a nucleic acid sequence encoding at least one Genome Editing Component (GEC) to produce a first maize plant wherein the genome of the maize haploid inducer comprises a supernumerary chromosome, and wherein the at least one GEC is introduced into the supernumerary chromosome;
b) providing a second maize plant, wherein the second maize plant comprises the maize plant genomic DNA which is to be edited;
c) crossing the second maize plant with the first maize plant;
d) selecting at least one haploid progeny produced by the cross of step (c) wherein the haploid progeny comprises the genome of the second maize plant and the supernumerary chromosome from said first maize plant but otherwise lacks the genome of the first maize plant;
e) crossing the progeny from step (d) with itself or a third maize plant; and
f) selecting offspring from the progeny of step (e) wherein the progeny lacks the supernumerary chromosome from said first plant and comprises the at least one targeted genome modification.
16. The method of claim 1 , the method further comprising:
e) crossing the progeny from step (d) with itself or a third maize plant; and
f) selecting offspring from the progeny of step (e) wherein the progeny lacks the supernumerary chromosome from said first plant and comprises the at least one targeted genome modification.