Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9.

Area of Science:

• Plant genetics
• Molecular biology
• Immunology

Background:

• Coeliac disease is an autoimmune disorder triggered by gluten ingestion in genetically susceptible individuals.
• Wheat α-gliadin peptides, particularly the 33-mer, are key immunodominant triggers in coeliac disease patients.
• Genetic modification offers a potential strategy to reduce immunogenic gluten components in wheat.

Purpose of the Study:

• To develop genetically modified wheat lines with significantly reduced levels of immunogenic α-gliadin peptides.
• To utilize CRISPR-Cas9 technology for targeted gene editing of α-gliadin genes.
• To assess the efficacy and specificity of gene editing in reducing gluten content and immunoreactivity.

Main Methods:

• Design and application of two single-guide RNAs (sgRNAs) targeting conserved regions of α-gliadin genes.
• Generation of mutant wheat lines using CRISPR-Cas9 gene editing.
• Analysis of α-gliadin content, gene mutation status, and immunoreactivity in generated lines.
• Screening for transgene-free lines and assessment of potential off-target mutations.

Main Results:

• Twenty-one mutant wheat lines were successfully generated, all exhibiting a substantial reduction in α-gliadins.
• One line showed mutations in up to 35 different α-gliadin genes out of 45 identified in wild-type wheat.
• Immunoreactivity was reduced by 85% in the mutant lines.
• Transgene-free lines were identified, and no off-target mutations were detected in potential target sites.

Conclusions:

• The developed low-gluten, transgene-free wheat lines demonstrate a significant reduction in immunogenic α-gliadins.
• These lines hold promise for producing low-gluten foodstuffs for individuals with coeliac disease.
• The technology provides a valuable source for introgressing low-gluten traits into elite wheat varieties.