First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible induce point mutations in one or some target sequences simultaneously, well introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored forest species. In study, we reported first example of CRISPR/Cas9-mediated gene editing Castanea genus. As a proof concept, targeted encoding phytoene desaturase ( pds ), whose mutation disrupts chlorophyll biosynthesis allowing visual assessment knockout Globular early torpedo-stage somatic embryos sativa (European chestnut) were cocultured 5 days with construct targeting two conserved regions subsequently cultured on selection medium kanamycin. After 8 weeks subculture medium, four kanamycin-resistant embryogenetic lines isolated. Genotyping these through Sanger sequencing amplicons revealed successful editing. Cotyledonary maturated maltose 3% cold-stored at 4°C 2 months. Subsequently, subjected germination process produce albino plants. study opens way use system European chestnut biotechnological applications