Engineering ultra-low-gliadin wheat for celiac disease using an integrated RNAi, CRISPR, and doubled haploid strategy.

The growing prevalence of gluten-related disorders has driven the development of wheat varieties with reduced immunogenic gluten. This study aimed to integrate RNA interference (RNAi) and CRISPR genome editing within a doubled haploid (DH) platform to overcome challenges of gene redundancy and polyploidy in wheat gliadins. We generated DH lines from crosses between RNAi and CRISPR lines and elite wheat cultivars, enabling stable fixation of multiple genetic modifications in a single generation. Deep sequencing analysis of α-gliadin amplicons was conducted using a custom bioinformatics pipeline optimized for complex, repetitive gene families. Gluten protein profiles were evaluated using RP-HPLC and R5 monoclonal antibody. Several DH lines presented over 70% reduction in immunogenic epitopes in α-gliadins, with lines outperforming both parents. Editing frequency was influenced by single-guide RNA efficiency and parental background. Silencing and editing combined led to nearly depleted gliadins in some lines, often with compensatory increases in other storage proteins linked with bread-making quality, such as high-molecular-weight glutenin subunits. Kernel and specific weight traits were largely maintained. This work demonstrates that combining RNAi and CRISPR in a DH platform enables efficient, heritable reduction of immunogenic gluten, providing a viable strategy for breeding wheat lines safer for individuals with gluten-related disorders.