The advent of genome editing technologies such as CRISPR-Cas9 has revolutionized the development of disease-resistant crops, offering precision and efficiency in targeting specific genetic loci responsible for susceptibility. In this protocol, we harness the CRISPR-Cas9 system to disrupt key susceptibility genes in tomatoTomato, aiming to fortify resistance against tobamoviruses, particularly the aggressive tomato brown rugose fruit virus (ToBRFV). By systematically knocking out four TOM1 homologs, genes essential for tobamoviral replication, tomato lines with robust and heritable resistance can be developed while minimizing adverse developmental effects. The approach not only underscores the significance of basic research on host—pathogen interactions in modern crop protection but also lays the groundwork for sustainable, gene-driven resistance strategies in commercial tomato breeding.