Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families

Abstract

Background Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1–2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. Results In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. Conclusions The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods. Electronic supplementary material The online version of this article (10.1186/s12870-019-1889-5) contains supplementary material, which is available to authorized users.

Recent Progress and Recommendations on Celiac Disease From the Working Group on Prolamin Analysis and Toxicity

“…This line showed a 85% reduction of the R5 and G12 ELISA signals. Jouanin et al (150) simultaneously edited multiple sites in α-and γ-gliadins with a single construct. Although the lines produced in these pilot studies are not yet safe, they demonstrate the power of gene editing for effectively modifying tens of genes of multiple gene families in a polyploid species at once.…”

Status and Contribution of CRISPR/Cas9 Based Gene-Editing System in the Development of a Low-Immunogenic Wheat Variety

“…Among all these, CRISPR/cas9 is the latest gene-editing tool that is highly accurate, rapid, simple and comparatively cheaper than other gene-editing tools [27,28]. CRISPR/Cas9 system is successfully applied for plant genome improvement and in various human diseases such as gastrointestinal, hematologic, viral, and cancer [9,29].…”

“…While gliadin makes the dough soft, glutenins provide a fine baking quality to wheat [8]. Gliadin is sub-divided into α-, γ-, and ω-subfractions, out of which α-gliadin contains critical epitopes responsible for CD development [9]. There are two fractions of glutenins i.e., low molecular weight (LMW) and high molecular weight (HMW) glutenins [8].…”

“…There are two fractions of glutenins i.e., low molecular weight (LMW) and high molecular weight (HMW) glutenins [8]. Gliadin is encoded by multiple gene families that are arrayed on specific loci in a repetitive sequence fashion and are located at Gli-2 loci on chromosome 6A, B and D [2,9]. α-gliadin contains the 33-mer peptide, particularly rich of proline-glutamine sequences.…”

“…CRISPR/Cas9 is a novel secondgeneration genome editing system. This technique can apply in wheat to efficiently modify qualitatively and/or quantitatively the gliadin content in wheat grains [9]. CRISPR/cas9 system targets a particular genome locus and delete a desired gene within the genome, this machinery may use in the wheat to replace the toxic gluten gene with a preferred lowimmunogenic gene to produce gluten-free/low gluten-containing wheat.…”