Reduced activity of an immunogenic macromolecule Amylase Trypsin inhibitor (ATI) in wheat through CRISPR/Cas9 mediated multiple gene editing

Wheat is the staple food for 40% of the world, providing 20% of dietary energy and protein. However, along with providing nutrition, wheat contains several anti-nutritional macromolecules. Amylase/Trypsin inhibitors (ATIs) are one such macromolecular proteins which have been known to cause allergic reactions like baker's asthma, auto-immunogenic reactions like Non-Celiac Wheat Sensitivity, and primary triggers for Celiac Disease in some predisposed humans. Bread wheat varieties without ATI molecules or with reduced activity have not yet been developed. Here, multiple genes of major ATI protein molecules were mutated using tRNA-based multiplex CRISPR/Cas9 genome editing technology. ATI proteins were extracted from wheat flours of gene-edited wheat lines along with unedited plants and subjected to quantification, detection by SDS-PAGE, fractionation by HPLC, and assayed the α-amylase and trypsin inhibition activity. Gene-edited Bobwhite wheat plant produced seeds with reduced (up to 30.61%) ATI content, which resulted in a reduction in α-amylase and trypsin inhibition activity to 50.74% and 44.90%, respectively. Another variety of bread wheat HD2967 also showed a significant reduction in ATIs content as well as a reduction in α-amylase and trypsin inhibition activity. This result suggests the possibility of developing low immunogenic wheat lines by multiple gene editing for the immunogenic macromolecules.