CRISPR/Cas multiplex gene editing of gamma and omega gliadins, paving the way for gluten-free wheat
Wheat is a staple cereal in the human diet, providing 20% of the calories we need daily. Despite its significance, the consumption of wheat-containing products is prohibited for an increasing percentage of the population suffering adverse reactions to wheat. These reactions are triggered by wheat gluten, particularly the gliadin fraction, whose DQ2.5-restricted epitopes elicit an immune response in individuals with celiac disease. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into the genes of wheat gamma- and omega-gliadins, providing wheat lines deficient in one or both immunogenic gliadin fractions simultaneously. To that, 8 guide RNAs were designed and combined into 4 plasmids to produce 54 lines, of which 20 exhibited mutations in the target gliadin genes. Characterization of these lines through Sanger or NGS sequencing, RP-HPLC, and monoclonal antibodies revealed a complex pattern of InDels, including deletions spanning multiple guide RNAs, as well as small InDels or insertions at the cleavage sites. When multiple RNA guides hit the same gliadin gene, it is possible to extract the fragment between them. The mutations were transmitted to the offspring, and the analysis of homozygous-derived lines showed a 97% reduction in gluten content. The targeted mutations and reduction of gliadins led to a compensatory effect, increasing the grain glutenin fractions. Crossbreeding these lines with other CRISPR/Cas lines deficient in the alpha-gliadins allowed mutations to be combined, resulting in lines with very low gluten content. This work represents an important step forward in the CRISPR/Cas development of wheat lines lacking immunogenicity.