Zitong Yu, Ural Yunusbaev, Allan Fritz, Michael Tilley, Alina Akhunova, Harold Trick, Eduard Akhunov

Plant Biotechnology Journal; First published: 17 November 2023;  https://doi.org/10.1111/pbi.14231

Summary

Wheat 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.

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14231

Figure 2: Summary of gene editing events identified by whole genome sequencing in the ω- and γ-gliadin gene clusters. (a, b) The distribution of the depth of read coverage, the types of editing events, and gRNA target sites with 0, 1, 2 or 3 mismatches to the gRNA protospacers for the ω-gliadin genes with the deletion of a cluster of three genes (a) and short deletions in the coding region (b). (c) Distribution of gRNA target sites and different types of gene editing events among the ω- and γ-gliadin genes on chromosomes 1A, 1B and 1D.