Insights from CRISPR/Cas9-mediated gene editing of centromeric histone H3 (CENH3) in carrot (Daucus carota subsp. sativus)

The generation of haploids is one of the most powerful means to accelerate the plant breeding process. In most crop species, an efficient haploid technology is not yet available or only applicable to a limited set of genotypes. Recent results published for Arabidopsis thaliana and major cereal crops like maize and wheat about successful haploid induction by CRISPR/Cas9-mediated editing of the centromeric histone H3 gene (CENH3) suggest that this novel method for the production of haploid plants might also be applicable to vegetable species like carrot. Here, we report and summarize the different experimental and genetic approaches that have been focused in the past few years on CRISPR/Cas9-based editing of the carrot CENH3 gene. We also describe the discovery of a second CENH3 locus in the carrot genome, which complicates the attempts to generate and to analyse putative haploid inducer genotypes. We show that three different CRISPR/Cas9 target constructs, used alone or in combinations, could successfully target carrot CENH3. Promising mutants such as in-frame indel or in-frame deletion mutants have been found, but their successful usage as putative haploid inducer is uncertain yet. Next generation sequencing of amplicons spanning CRISPR target sites and transcript-based amplicon sequencing seemed to be appropriate methods to select promising mutants, to estimate mutation frequencies, and to allow a first prediction which gene was concerned. Another aim of this study was the simultaneous knockout and complementation of the endogenous carrot CENH3 gene by an alien CENH3 gene. Co-transformation of a CRISPR/Cas9-based carrot CENH3 knockout construct together with a CENH3 gene cloned from ginseng (Panax ginseng) was performed by using Rhizobium rhizogenes. It was shown, that ginseng CENH3 protein is accumulated inside the kinetochore region of carrot chromosomes, indicating that PgCENH3 might be a suited candidate for this approach. However, presently it is unclear, if this gene is fully functioning during the meiotic cell divisions and able to complement lethal gametes. Challenges and future prospects to develop a CENH3-based HI system for carrot are discussed.