Genome editing and regeneration pipeline for engineering disease resistance in tomato using CRISPR/Cas9

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9)-based genome editing has emerged as a powerful tool for developing disease-resistant crops. Here, we present a comprehensive and reproducible protocol for applying CRISPR/Cas9 genome editing in tomato (Solanum lycopersicum), covering guide RNA (gRNA) design using CRISPOR, Golden Gate vector assembly, Agrobacterium-mediated transformation, plant regeneration, and molecular validation of edited plants. The workflow integrates standardized bioinformatics and sequencing-based validation tools, including DSDecodeM, TIDE, and protein-level impact analysis, to confirm targeted mutations and assess editing efficiency. Quantitative benchmarks for regeneration, transformation, and editing efficiencies were provided to support reproducibility. This protocol offers an integrated pipeline for generating and validating targeted gene knockouts in tomatoes and is intended to facilitate functional genomic studies and the development of disease-resistant cultivars. However, it is more widely applicable to gene editing in tomato plants.