CRISPR-based editing of the omega- and gamma-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality

Authors: YU, ZITONG - Kansas State University; YUNUSBAEV, URAL - Kansas State University; FRITZ, ALLAN - Kansas State University; Tilley, Michael - Mike; AKHUNOVA, ALINA - Kansas State University; TRICK, HAROLD - Kansas State University; AKHUNOV, EDUARD - Kansas State University

Submitted to: Nature Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/30/2023
Publication Date: 4/1/2024
Citation: Yu, Z., Yunusbaev, U., Fritz, A., Tilley, M., Akhunova, A., Trick, H., Akhunov, E. 2024. CRISPR-based editing of the omega- and gamma-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality. Nature Plants. https://doi.org/10.1111/pbi.14231.
DOI: https://doi.org/10.1111/pbi.14231

Interpretive Summary: The gluten proteins are the primary determinants of end-use quality in wheat and are largely responsible for controlling dough quality characteristics. Gluten is composed of 2 complex protein groups, gliadin and glutenin. Certain susceptible individuals have adverse reactions to wheat gluten and must avoid products made from wheat. Different types of gliadin proteins contain sequences that lead to a toxic immunological response. Attempts to reduce the immunotoxicity of gluten using breeding and biotechnology often affect dough quality due to the large number of gluten proteins. Thus, wheat that is safe for sensitive individuals but retains baking quality cannot be produced by conventional breeding alone. CRISPR/Cas9 is a tool that allows targeted editing of specific genes. CRISPR-Cas9 was used to modify the immunoreactive peptides of gliadin genes. The edited wheat lines showed a 50% to 60% reduction in gliadin content and had no negative impact on grain protein quality.

Technical Abstract: Wheat gluten defines the breadmaking properties of dough and underlies gluten sensitivity to wheat-based products. Gluten is composed of 2 protein types, gliadins and glutenins. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality due to the large number of gluten proteins. Thus, wheat that is safe for sensitive individuals but retains baking quality cannot be produced by conventional breeding alone. CRISPR/Cas9 is a tool that allows targeted editing of specific genes. CRISPR-Cas9 was used to modify gliadin genes in the immunoreactive peptides identified in the genomes assembled using the long-read sequencing technologies. The whole genome sequencing of an edited line showed editing or deletion of nearly all gamma-gliadin and half of the omega-gliadin gene copies and lack of editing in the alpha/beta-gliadin genes. The detected 62% and 52% reduction in omega- and gamma-gliadin content, respectively, had no negative impact on grain protein quality. A 47-fold immunoreactivity reduction compared to wild type was detected using antibodies against immunotoxic peptides. Our results indicate that genome profiling to identify gliadin gene copies abundant in immunoreactive peptides and their targeted editing could be an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact on the end-use quality traits.