Development of virus-induced genome editing methods in Solanaceous crops.

Genome editing (GE) using CRISPR/Cas systems has revolutionized plant mutagenesis. However, conventional transgene-mediated GE methods have limitations due to the time-consuming generation of stable transgenic lines expressing the Cas9/single guide RNA (sgRNA) module through tissue cultures. Virus-induced genome editing (VIGE) systems have been successfully employed in model plants, such as and spp In this study, we developed two VIGE methods for Solanaceous plants. First, we used the (TRV) vector to deliver sgRNAs into a transgenic tomato () line of cultivar Micro-Tom expressing . Second, we devised a transgene-free GE method based on a (PVX) vector to deliver and sgRNAs. We designed and cloned sgRNAs targeting in the VIGE vectors and determined optimal conditions for VIGE. We evaluated VIGE efficiency through deep sequencing of the target gene after viral vector inoculation, detecting 40.3% and 36.5% mutation rates for TRV- and PVX-mediated GE, respectively. To improve editing efficiency, we applied a 37°C heat treatment, which increased the editing efficiency by 33% to 46% and 56% to 76% for TRV- and PVX-mediated VIGE, respectively. To obtain edited plants, we subjected inoculated cotyledons to tissue culture, yielding successful editing events. We also demonstrated that PVX-mediated GE can be applied to other Solanaceous crops, such as potato () and eggplant (). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.