Development of virus-induced genome editing methods in Solanaceous crops

Abstract

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 <i>Arabidopsis thaliana</i> and <i>Nicotiana</i> spp<i>.</i> In this study, we developed two VIGE methods for Solanaceous plants. First, we used the <i>tobacco rattle virus</i> (TRV) vector to deliver sgRNAs into a transgenic tomato (<i>Solanum lycopersicum</i>) line of cultivar Micro-Tom expressing <i>Cas9</i>. Second, we devised a transgene-free GE method based on a <i>potato virus X</i> (PVX) vector to deliver <i>Cas9</i> and sgRNAs. We designed and cloned sgRNAs targeting <i>Phytoene desaturase</i> 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 (<i>Solanum tuberosum</i>) and eggplant (<i>Solanum melongena</i>). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.