Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9
Sanchez-Leon, Susana; Gil-Humanes, Javier; Ozuna, Carmen V.; Gimenez, Maria J.; Sousa, Carolina; Voytas, Daniel F.; Barro, Francisco
Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The alpha-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 alpha-gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in alpha-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.
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