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.

Keywords: CRISPR/Cas9; Coeliac disease; Gene editing; Gluten; Mutation breeding; Polyploid; Wheat; α-Gliadin; γ-Gliadin; γ-Irradiation.

Fig. 1

Alignment of α- and γ-gliadin protein sequences with sgRNA position and potential sites of gene editing. A representation of the protein sequence alignments of α-gliadins (a) and γ-gliadins (b) based on a total of 438 and 187 DNA sequences, respectively. The variation in the sequences form patterns which are grouped here and associated to the genome in which they are mostly found (A, B, D on the left side of each group), based on comparison of hexaploid wheat sequences with sequences from diploid relatives. The different gliadin protein domains are indicated and the position of the CD immunogenic epitopes are boxed. The DQ2.5 epitopes box includes the DQ2.5-α1, − α2 and - α3 epitopes (Additional file 1: Figure S1 and Additional file 2: Figure S2). The sgRNAs targeted motifs are highlighted in yellow and the potential gene editing sites are marked with scissors. sgRNA_γ272 may cut multiple times, depending on the number of repetitions of the most abundant γ-gliadin CD epitope, DQ2.5-glia-γ4c, which it targets. Details on the alignment, sequence patterns and CD epitopes can be found in Additional file 1: Figure S1 and Additional file 2: Figure S2. The MEGA and fasta files are also provided as Additional Files.

Fig. 2

Representation of one α-gliadin Gli-2 locus and different mutation types potentially induced by CRISPR/Cas9. This schematic α-gliadin Gli-2 loci representation shows genes clustered and different types of mutations that can be induced by sgRNA. Simultaneous cuts in non-consecutive genes can delete the intervening genes. Similarly, two simultaneous cuts flanking the epitope can delete only this region, whilst simple small indels or base substitutions can also occur.

Fig. 3

Acid-PAGE of Chinese Spring deletion lines showing altered endosperm gliadin protein profiles. Gliadin extracts from grains of Chinese Spring nullisomic/tetrasomic lines and Kansas deletion lines were run on non-denaturing Acid-PAGE alongside a gliadin extract from Chinese Spring (CS WT). The lanes displayed next to each other have been run alongside each other on the same gel but each panel represents a different gel. Each sample was always run alongside CS WT as a control. The black and grey arrows point at the changes in the protein groups from the deleted chromosome arms and in others respectively. CS gliadin profile in absence of a Chr1-AS, b Chr1-BS, c Chr1-DS, d part of Chr6-AS, e Chr6-DS, f part of Chr1-BS and Chr6-DS.

Fig. 4

Acid-PAGE of selected Paragon γ-irradiated mutant lines that showed changes in gliadin protein profiles. Gliadin extracts from grains of the M4 generation of Paragon γ-irradiated mutant population were run on non-denaturing Acid-PAGE alongside a gliadin extract from Paragon (Paragon WT). Each panel represent a different gel. The lanes displayed next to each other have been run alongside each other. The black arrows point at the changes observed in the irradiated lines. a and b α-gliadin bands missing, probably correlated to changes in Chr6-AS, c and d α-gliadin bands changes that are different from any change observed in deletion lines and nullisomic/tetrasomic lines, e γ- and ω-gliadin bands missing, probably due to changes in 1BS, f γ-gliadin bands missing and ω-gliadin bands shifts and intensity changes, probably due to changes in Chr1-AS.

Fig. 5

Acid-PAGE of T1 grains showing altered gliadin protein profiles. Gliadin extracts from Fielder-CRISPR T1 grain from each of the 4 constructs were run on non-denaturing Acid-PAGE alongside the gliadin extract from Fielder wild type. Each panel represent a different gel. The lanes displayed next to each other have been run alongside each other. The start of the sample names refers to the constructs with the sgRNAs they include (α1, α2, γ3 or α2γ3), followed by the T0 plant line and grain number. The black and grey arrows point respectively at the changes intended or unintended by the construct present in the plant that set the grains. a α-gliadin bands missing likely related to mutations on Chr6-AS, b α-gliadin bands missing or lower expressed likely related to mutations on Chr6 in all 3 homoeologous genomes, c ω-gliadin bands shifted up and γ-gliadin band with lower expression level likely related to mutations on Chr1-BS or DS, d ω- γ- and α-gliadin bands with lower expression likely related to mutations on Chr6-AS, e) ω-gliadin bands shifted up and γ-gliadin bands missing likely to be related to Chr1-AS, similar mutant profile in two different T1 grains from the same T0 plant.

Fig. 6

CRISPR/Cas9 T-DNA construct 2α _sgRNA. Construct 2α_sgRNA that contains sgRNA_ α213 and sgRNA_ α324, as an example of the four T-DNA constructs generated. They are similar, only the number and nature of sgRNA integrated are different.