Cas9- and Cas12a-mediated excision and replacement of the celiac disease-related α-gliadin immunogenic complex in hexaploid wheat.

Celiac disease (CD) is a chronic enteropathy affecting approximately 1% of the global population. Wheat α-gliadins are a major contributor to the autoimmune response, as they contain one of the most immunogenic peptides, the 33-mer, along with numerous variants. In this study, we used CRISPR/Cas technology to mutate genes encoding α-gliadins. This approach employed paired sgRNAs to precisely excise immunogenic regions while preserving non-immunogenic sequences within the α-gliadins. Furthermore, we replaced the excised region with an α-gliadin-based double-stranded oligodeoxynucleotide (dsODN) designed with nucleotide changes to reduce immunoreactivity and increase peptidase cleavage sites. Two different CRISPR systems, Cas9 and Cas12a, were applied to generate wheat protoplasts and plants with non-immunogenic regions. Cas9 demonstrated superior performance in terms of editing frequency, excision and replacement of immunogenic fragments. However, the Cas12a nuclease (Cpf1) showed promising editing efficiency, offering the potential for future wheat editing applications. Using the Cas9 system, we achieved a 74.2% excision rate of the 33-mer in wheat plants. Subsequent analyses showed a significant reduction in the reactivity to the G12 monoclonal antibody, capable of identifying the 33-mer peptide and a decrease in the prolamin levels compared to the wild-type. Additionally, we developed a high-throughput sequencing-based software specifically designed to identify mutations in multi-copy gene families. This innovative tool enabled fast, parallel screening of the samples in this study and facilitated the identification of the specific editing patterns produced by the designed constructs.