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. 2018 Apr;16(4):902-910.
doi: 10.1111/pbi.12837. Epub 2017 Nov 24.

Low-gluten, Nontransgenic Wheat Engineered With CRISPR/Cas9

Free PMC article

Low-gluten, Nontransgenic Wheat Engineered With CRISPR/Cas9

Susana Sánchez-León et al. Plant Biotechnol J. .
Free PMC article


Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α-gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33-mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33-mer in the α-gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in α-gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene-free lines were identified, and no off-target mutations have been detected in any of the potential targets. The low-gluten, transgene-free wheat lines described here could be used to produce low-gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.

Keywords: CRISPR/Cas9; coeliac disease; α-gliadins.


Figure 1
Figure 1
Gene editing of α‐gliadins in bread wheat. (a) Schematic of a typical α‐gliadin gene indicating the different protein domains. Two of the peptide sequences involved in gluten intolerance (p31‐43 and the 33‐mer) are represented by red arrows, whereas the target sequences for the sgRNAs (sgAlpha‐1 and sgAlpha‐2) are represented by blue arrows. Black arrows indicate primers used for Illumina sequencing. (b–d) Illumina sequencing of the α‐gliadin genes of 3 T1 BW208 mutant lines (T544, T545 and T553) transformed with sgAlpha‐2. (b) Alignment of the different deletion types found at the target locus of sgAlpha‐2; (c) Alignment of the different insertions at the target locus of sgAlpha‐2; and (d) frequency of the different type of insertions and deletions.
Figure 2
Figure 2
Characterization of sgAlpha‐1 and sgAlpha‐2 mutant plants. (a) A‐PAGE of gliadins from sg Alpha‐1 T1 half‐seeds (named as T566 and T567 lines) derived from T0 plant 14, and V323 and V343 (from T0 plant 77) and the corresponding wild‐type lines BW208 and THA53. Migration of α‐, γ‐, ω‐gliadin protein bands are outlined by brackets (b) MALDI‐TOF analysis of the same gliadin extract in (a) from T567 track and the BW208 wild type. Values are in absolute intensity. Left axis corresponds to T567 and the right axis to the BW208 line. The corresponding range of masses (m/z) for α‐, γ‐, ω‐gliadins are indicated by arrows. (c) A‐PAGE of gliadins from sgAlpha‐2 T1 half‐seeds (named as T544, T545 and T553 lines) from T0 plant 10, V491 and V496 (plant 17), T666, T668 and T670 (plant 2) and the wild‐type lines BW208, THA53 and DP. (d) MALDI‐TOF analysis of the same gliadin extracts in (a) from T553 track (plant 10) and T558 (plant 12, Figure S8), and the BW208 wild type. (e) Bar graph of fold change of α‐, γ‐, ω‐ and total gliadin fractions in bread and durum wheat transformed with sgAlpha‐1 and sgAlpha‐2. Values for each plant were normalized by values of the corresponding wild‐type lines. Note that A‐PAGE analysis is not a quantitative test, and intensity differences observed in the gels might be explained in part by differences in the amount of protein loaded and/or by differences in the staining/distaining process.
Figure 3
Figure 3
Analysis of Immune reactivity, SDS sedimentation volumes and gliadin profile of nontransgenic DP‐derived lines, and phenotype of sgAlpha‐derived lines. (a) Analysis of T2 seeds of the sgAlpha‐1 and sgAlpha‐2 mutant lines with the monoclonal antibodies (mAb) R5 and G12. Error bars, mean ± SD. Statistically significant differences between each mutant line and the wild type were denoted *< 0.05, **< 0.01 (Tukey HSD all‐pairwise comparisons test) (b) Sodium dodecyl sulphate (SDS) sedimentation test expressed as mLg‐1. T2 and T3 seeds from each line were bulked and three independent biological replications analysed. Error bars, mean ± SD. * Means are significantly different to wild types as determined by Dunnett's multiple comparisons at < 0.05. (c) Content of the omega, alpha and gamma‐gliadin fraction of the nontransgenic DP‐derived lines. Error bars, 5% Confidence Interval of the mean value of the wild‐type DP line. (d) A‐PAGE of gliadins from half‐seeds of the nontransgenic DP‐derived lines. Migration of α‐, γ‐, ω‐gliadin protein bands are outlined by brackets. (e) Spikes and seeds of sgAlpha‐2 BW208 mutant line in comparison with its wild type. (f) Spikes and seeds of sgAlpha‐2 DP mutant line in comparison with its wild type.

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