CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins, paving the way for gliadin-free wheat

Susana Sánchez-León, Miriam Marín-Sanz, María H Guzmán-López, Marta Gavilán-Camacho, Edurne Simón, Francisco Barro

Journal of Experimental Botany, erae376, https://doi.org/10.1093/jxb/erae376

Abstract

Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this work, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger or NGS sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by RP-HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.

See https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/erae376/7750082?login=false

Figure 2: (A)-PAGE gliadin profiles of edited wheat lines. A-PAGE gliadin profiles of the offspring of (A) T0 lines transformed with pSSLGamma16, (B) pSSLOmega8, and (C) pSSLOmega9. The red arrows and red boxes indicate the absence of bands or softer ones, and the green arrows indicate new bands in the edited lines.