Development of an efficient and heritable virus-induced genome editing system in <i>Solanum lycopersicum</i>

The CRISPR-Cas9 system can be used to introduce site-specific mutations into the genome of tomato (<i>Solanum lycopersicum</i>) plants. However, the direct application of this revolutionary technology to desirable tomato cultivars has been hindered by the challenges of generating transgenic plants. To address this issue, we developed an efficient and heritable genome editing system using tobacco rattle virus (TRV) for an elite tomato cultivar (the paternal line of Saladette). Notably, this virus-induced genome editing (VIGE) system enables the rapid production of various mutant seeds without the need for additional plant transformation and tissue culture, once a Cas9-expressing tomato line is established. This VIGE system consists of transgenic tomato plants that express Cas9 under the control of the tomato ubiquitin 10 (<i>SlUbi1</i>0) gene promoter and a mobile guide RNA scaffold (gRNA:SlmFT) generated using the sequence of the tomato <i>Flowering Locus T</i> (<i>SlFT</i>) gene. We determined its editing efficiency by targeting the tomato phytoene desaturase (<i>SlPDS</i>) gene, which causes photobleaching symptoms when disrupted. Most transgenic seedlings infected with the TRV vectors carrying the <i>SlPDS-</i>targeting sgRNA developed chimeric albino leaves associated with a high frequency of indel mutations in the <i>SlPDS</i> gene. Remarkably, fruits from these plants yielded homozygous <i>SlPDS</i> knockout seeds at rates ranging from 15% to 100%. These results demonstrate the exceptional effectiveness of our VIGE system in rapidly generating heritable genome edits in tomato.