Tomato brown rugose fruit virus (ToBRFV) poses a major threat to global tomato (Solanum lycopersicum) production, as it can overcome conventional resistance genes that are effective against tobamoviruses. In this study, a multiplex CRISPR/Cas9 system was developed to target the SlTOM1 susceptibility gene family (SlTOM1a–d), which encodes host factors essential for tobamovirus replication. Six guide RNAs (gRNAs), designed following 12 off-target analyses, were assembled into a multiplex CRISPR/Cas9 construct using a Golden Gate cloning strategy and introduced into tomato genotypes through an Agrobacterium-based tissue culture transformation procedure. Although primary T0 transformants exhibited chimeric mutation patterns, stable inheritance and segregation of edited alleles were confirmed in the T1 generation. Sequence analyses identified diverse indel mutations across target loci, with SlTOM1d exhibiting the highest editing efficiency. Multiplex genome editing successfully generated single-, double-, and triple-mutant combinations, with higher-order mutants displaying the strongest tolerance phenotypes. Following mechanical ToBRFV inoculation, edited T1 plants exhibited markedly reduced symptom severity, low viral accumulation, and improved fruit health compared to wild-type controls. RT-qPCR analysis further confirmed significantly reduced viral RNA levels, supporting a host-factor-mediated tolerance mechanism. Importantly, edited lines maintained normal growth and agronomic performance. Collectively, these findings demonstrate that multiplex CRISPR/Cas9-mediated targeting of SlTOM1 homologs represents a promising and practical strategy for improving ToBRFV tolerance in tomato breeding programs.
Sarıkaya et al. (2026) studied this question.