Developing and evaluating CRISPR-Cas9 edited transgene-free soybeans with dramatic reduction in trypsin and chymotrypsin inhibition based on selfing and phenotyping T1 to T3 seeds

Soybeans (Glycine max) are a major source of plant proteins for food and feed globally. Still, the natural presence of Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) compromises their nutrition. In a previous study, the evolution of BBI genes was investigated, and a group of highly expressed seed-specific BBI genes in legumes were found. By using a CRISPR-Cas9 system targeting eight seed-specific BBI genes in soybeans, a hypothesis regarding the physiological role of BBI was validated, and mutants were generated with the drastic reduction in protease inhibition but little changes in agronomic traits. The present study aimed at developing transgene-free soybean mutants with as many homozygous edited BBI genes as possible by selfing segregation, genotyping T0-T2 plants, and phenotyping T1-T3 seeds for potential commercial applications. From T1 to T2 plants, homozygous mutations increased to five genes with different editing patterns, but T1-T3 seeds, either transgenic or transgene-free, all had drastic and consistent reductions in not only reactive BBI (79–82 %) but also reactive KTI (48–67 %) — an unanticipated change, leading to dramatic reductions in total trypsin inhibition (68–77 %) and total chymotrypsin inhibition (76–81 %) against a wild-type. On SDS-PAGE, mutant seeds exhibited no BBI band but a KTI band as intensive as the wild type. The observation indicated a possible disturbance of the reactive site or binding affinity of KTI to trypsin by significant reduction in BBI. Furthermore, a heating experiment demonstrated that based on BBI content and chymotrypsin inhibition, the mutant soybeans could bypass heating or at least shorten it by half, which is commonly required for conventional soybeans for optimal nutrition. Thus, the new soybeans were commercially valuable.