Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer

In the present study, maize plants are generated containing the Cas9 gene scissors and guide RNAs for target regions in two different genes in wheat. The target genes BRI1 and SD1 regulate the growth of wheat. The scientists use 5 transgenic maize plants for each target region in the wheat (targets determined by the guide RNA) and use them to pollinate the wheat plants. So it is a cross-species pollination. The maize chromosomes with the DNA encoding the gene scissors and respective guide RNAs are degraded in the course of the cell cycle, leaving behind the changes in the genetic material of the wheat. Since the corn’s chromosomes are broken down, there is only one set of chromosomes that comes from the wheat egg. To create a viable embryo, a technique called doubled haploidy is used. Here the duplication of the single set of chromosomes is generated by the use of a chemical substance called colchicine. The resulting plants are genetically stable. Such plants are called double haploids in contrast to normal diploid plants.

The resulting embryos are kept in the cell culture and wheat plants are regenerated. Subsequent investigations of the respective target region show that changes were generated by the gene scissors in all target regions and in all wheat lines used (summer wheat, winter wheat and pasta wheat). One of the strengths of the method described here is the work with double haploids, as homozygous wheat plants can be produced with the intended changes. It turns out, however, that heterozygous wheat plants are also generated in relation to the changes in the target region.

Comment: Here, both the time of the formation and the dose of the gene scissors play a decisive role. There is therefore still a need for optimization, but it should be kept in mind that with stronger dose of the gene scissors, non-target effects can also be increased in the genome of the wheat. The paper lacks an investigation of non-target effects.

According to the study, the wheat is free of transgenes, so no fragments of the DNA encoding the gene scissors should be found in the wheat. However, it is not clearly described whether the absence of gene scissors was checked. Even if homologous recombination has a low efficiency in higher plants, the incorporation of genetic material from maize into the genome of wheat cannot be completely ruled out. Therefore, this aspect should also be considered when analyzing non-target effects.