Methods and compositions for genome editing via haploid induction
A genome editing and genome technology, applied in the introduction of foreign genetic material using vectors, genetic engineering, plant genetic improvement, etc., can solve problems such as slowness and hardship
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Embodiment 1
[0127] Example 1. Genome modification using a haploid inducer genome as a GEC carrier chromosome.
[0128] Modification of the genome of a target plant can be performed by using a maternal haploid induced cross to transiently express a genome editing component (GEC) in the vicinity of the genome inherited from the target plant.
[0129] For example, Cas9 / The gRNA construct is transformed into the genome of a plant known for its ability to induce haploid progeny, such as a maize maternal haploid inducer containing a high oil phenotype such as CAUHOI (X Dong, X Xu, L Li, C Liu, X Tian, W Li, S Chen (2014) Mol Breeding 34:1147-1158). gRNAs can be designed to target a gene or locus in the genome of a target maize line, for example a knockout mutation of the waxy (Wx) gene sequence in the genome of maize line B73 (M Shure, S Wessler, NFedoroff, Cell (1983) 35:225-233). Cas9 can be operably linked to a promoter such as maize Ubi-1 (AH Christensen, RA Sharrock, PH Quail, Plant ...
Embodiment 2
[0140] Example 2. Modification of plant genomes using supernumerary chromosomes as GEC carrier chromosomes.
[0141] Genome editing can be produced in the genome of a target plant by crossing the plant with a haploid-inducing line containing a supernumerary chromosome, such as a B chromosome, containing an expression cassette encoding a GEC such as Cas9 and an associated gRNA system.
[0142] Similar to the transient expression of GEC from the inducer genome shortly before and / or during and / or after fertilization as described in Example 1, in this example the cells of the haploid inducer contain cells containing genes designed to target the target maize A supernumerary chromosome of a gene or locus of a strain such as a gRNA targeting the Wx gene in the B73 genome, such as the B chromosome. Cas9 can be operably linked to a promoter, such as maize Ubi-1, and gRNA can be operably linked to a gene that can be released in pollen cells and / or egg cells and / or zygotes shortly before...
Embodiment 3
[0150] Example 3. Haploid Induction Combined with Delivery of Exogenous Nucleic Acids into Recipient Lines
[0151] Transgenic traits can be delivered to plants of interest through the use of haploid induced crosses. Nucleic acid sequences encoding the transgene of interest ("donor transgene") and site-specific enzymes are introduced into HI maize plants by standard breeding or plant transformation techniques. The site-specific enzyme is operably linked to a promoter capable of expressing the GEC in the pollen cell, egg cell and / or zygote before, during or after fertilization.
[0152] The HI maize plant containing the donor transgene and the GEC is crossed as a male to a second maize plant and the GEC is transiently expressed shortly before, during or after fertilization. GEC translocates the donor transgene from the genome of the HI maize plant into the targeted location within the maternal genome of the egg cell prior to loss of the paternal nuclear genome.
[0153] Induc...
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