The DELITE project aims to implement a new concept for the delivery of RNA-guided Cas endonucleases, which involves the use of plant lines capable of inducing the formation of haploid progeny when used as male parent. There are a number of established systems facilitating the formation of maternal haploids via cross-pollination followed by uniparental genome elimination. These include wheat x maize, barley x Hordeum bulbosum and maize x phospholipase A1 knockout (pla1A-KO) maize.
Upon genetic transformation of such haploidy-inducing lines using guide-(g)RNA and Cas expression units, respective transcript and ribonucleoprotein is produced and transferred in their sperm via pollination and fertilization to hybrid zygotes formed on the female parent. While the genome contributed to the zygote by this plant is now subjected to targeted modification, the transgene-carrying genome derived from the haploidy inducer is being completely eliminated in the context of the mitoses taking place in the early zygotic embryo that thereby becomes haploid. Resultant haploid progeny carrying site-specific modifications can be chemically induced to undergo whole genome duplication, which entails the formation of entirely homozygous plants. In the DELITE project, we envisage
to genetically transform Hordeum bulbosum and pla1A-KO maize line RWK using barley and maize target gene-specific gRNA/Cas constructs, respectively. The transgenic, haploidy-inducing lines will then be used to pollinate barley and maize in order to trigger site-directed modifications in these species. The approach pursued has several advantages over current methods.

(1) Site-directed alterations may be achieved in an unprecedented variety of genotypes including elite germplasm, since the use of haploidy-inducing lines is considered far less genotype dependent as compared with conventional methods of plant regeneration from transiently or stably transformed cells.

(2) Once a gRNA/Cas-transgenic haploidy inducer plant is available for a given target gene, it can be readily utilized
for targeted genome modification even in breeders' facilities, where haploid and genotyping technology is typically established.

(3) As cross-pollinations can be performed at virtually unlimited scope, just a single gRNA/Cas-transgenic haploidy
inducer plant is sufficient to generate a whole variety of mutant lines.

(4) The principle is associated with reduced modification of off-targets, because the presence of gRNA and Cas is confined to the period of early embryogenesis.

(5) Thanks to the elimination of the discrete, transgene-carrying genome from the developing embryo, the site-specifically modified genome of the produced plants is kept free from any transgenes during the whole process. (6) The preliminarily haploid genome of these plants is identically duplicated, thus homozygous individuals can be instantly obtained whose genetic modification will be genetically fixed as well.