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

Authors: Liu, Keshun; WANG, ZHIBO - Donald Danforth Plant Science Center; An, Yong Qiang

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2025
Publication Date: 3/20/2025
Citation: Liu, K., Wang, Z., An, Y. 2025. Developing CRISPR-Cas9 edited transgene-free soybeans with dramatic reduction in trypsin and chymotrypsin inhibition based on selfing and phenotyping T1 and T3 seeds. Journal of Agricultural and Food Chemistry. 22. Article 101811. https://doi.org/10.1016/j.jafr.2025.101811.
DOI: https://doi.org/10.1016/j.jafr.2025.101811

Interpretive Summary: Soybeans have long emerged as a major oilseed crop, providing oil and proteins for food, feed, and various industrial applications. Yet, the natural occurrence of Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) of trypsin and chymotrypsin in soybean seeds has long limited soybean utilization, because both trypsin and chymotrypsin are digestive proteases, and their inhibitors interfere with protein digestion for animals. In the United States and elsewhere, most of the soybean production is crushed into oil and defatted meal. The latter is primarily used as animal feed and contains about 48% proteins. Soymeal is typically subjected to a moist toasting process to inactivate the two protease inhibitors. Yet, the process not only increases the operation cost but also can reduce the nutritional value of soymeal when it is not optimally controlled. Therefore, scientists have long pursued an alternative by breeding soybeans with reduced protease inhibitors. So far, genetic means to reduce soybean trypsin inhibition have primarily focused on KTI reduction. Yet, the most recent work by scientists at the USDA-ARS showed that BBI inhibits chymotrypsin ten times stronger and trypsin two times stronger than KTI. This finding highlights the importance of targeting BBI for developing soybeans with reduced protease inhibition. Consequently, USDA-ARS scientists, along with those at Danford Plant Science Center, used Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 (CRISPR-Cas9), an emerging and efficient genome editing technique, to knockout several seed specific BBI genes in soybeans to reduce protease inhibitory activity in soybeans. Mutant soybeans with drastic reduction not only in BBI but also in KTI were developed, resulting in drastic reductions (70-80%) in protease inhibition. More importantly, the new soybean lines were selected for transgene-free (non GMO), with stable edited traits and no changes in key agronomic traits. This reported work represents a significant breakthrough in the development of soybeans that can bypass or at least reduce heating time by half while maintaining maximum nutritional values. The new soybeans have great potential for commercialization, benefiting the soybean industry and related industries, such as livestock, aquaculture, food and feed industries.

Technical Abstract: 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.