CRISPR/Cas9-Targeted Gene Editing of Allergenic Profilin-Encoding Lyc e1 in Tomato Fruit

Tomato (Solanum lycopersicum L.), one of the most widely consumed horticultural crops worldwide, is also a notable source of food allergens, with a higher prevalence of allergic reactions in pollen-sensitized patients. Profilin, a small actin-binding protein, is a major allergen in tomato fruits encoded by two homologous genes, Lyc e1.01 and Lyc e1.02. Although various strategies have been proposed to reduce allergenicity in foods, no prior study has successfully achieved the complete elimination of profilin proteins in tomato using precise genome editing. In this study, we designed a single-guide RNA targeting a conserved region of both genes and employed the CRISPR/Cas9 system to generate loss-of-function mutations. We first evaluated single-guide RNA editing efficiency in tomato protoplasts and then performed Agrobacterium-mediated stable transformation, which yielded 13 transgenic T₀ lines. Genotyping and Western blot analyses confirmed successful editing at both target loci and significantly decreased profilin accumulation in mutant tomato fruits. Notably, two homozygous Cas9-free lines were identified in the T1 generation, and one of these (line 23-15) showed significantly decreased profilin protein levels in the fruit. These findings demonstrate that the CRISPR/Cas9-mediated disruption of allergenic Lyc e1 genes effectively removes profilin from tomato fruits. This strategy provides a promising framework for developing hypoallergenic tomato cultivars that may be extended to other related crop species.