CRISPR‐based editing of the ω‐ and γ‐gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality

Abstract

SummaryWheat immunotoxicity is associated with abnormal reaction to gluten‐derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR‐Cas9 editing of cultivar Fielder was used to modify gluten‐encoding genes, specifically focusing on ω‐ and γ‐gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long‐read sequencing technologies. The whole‐genome sequencing of an edited line showed mutation or deletion of nearly all ω‐gliadin and half of the γ‐gliadin gene copies and confirmed the lack of editing in the α/β‐gliadin genes. The estimated 75% and 64% reduction in ω‐ and γ‐gliadin content, respectively, had no negative impact on the end‐use quality characteristics of grain protein and dough. A 47‐fold immunoreactivity reduction compared to a non‐edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR‐based modification of the ω‐ and γ‐gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high‐quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.