Computationally Validated CRISPR/Cas9-Mediated Genome Editing of the Acetolactate Synthase Gene for Herbicide Resistance in Tomato (Solanum lycopersicum Mill.)

Precise genome editing technologies offer powerful opportunities for crop improvement through targeted modification of agronomically important genes. In the present study, the efficiency of the CRISPR/Cas9 system was evaluated for inducing herbicide resistance in tomato (Solanum lycopersicum L.) by targeting the acetolactate synthase (ALS) gene, a key enzyme in the biosynthesis of branched-chain amino acids and a known target of sulfonylurea herbicides. A 19-nucleotide guide RNA (gRNA) targeting a conserved catalytic domain in exon 2 of the ALS gene was designed and cross-validated using CHOPCHOP v3 and CRISPOR algorithms, confirming high specificity (scores 97.6 and 94/97, respectively) with zero perfect-match off-targets in the tomato genome. An optimized in vitro regeneration protocol was established using cotyledon explants, with the highest shoot regeneration frequency (59.78%) obtained on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) and 0.5 mg L⁻¹ α-naphthaleneacetic acid (NAA). Agrobacterium-mediated transformation was performed using a CRISPR/Cas9 binary vector carrying the validated guide RNA specific to the ALS gene. Hygromycin selection yielded twenty-one putative transgenic lines, of which twelve T₀ plants were confirmed by PCR for the presence of both Cas9 and gRNA cassettes. Sequence analysis of the targeted ALS locus identified site-specific nucleotide substitutions in four T₀ lines, corresponding to a mutation efficiency of 19.05%. The detected mutations—comprising A→G transitions and A→C transversions—were located 15 bp downstream from the protospacer adjacent motif, consistent with CRISPR/Cas9-mediated editing and validating computational predictions. These results demonstrate the successful induction of targeted ALS gene mutations in tomato through computationally validated guide RNA design and establish a foundation for the development of herbicide-resistant lines, with potential applicability to future transgene-free genome editing strategies.