Kim, W.-S.; Gillman, J.D.; Kim, S.; Liu, J.; Janga, M.R.; Stupar, R.M.; Krishnan, H.B. Bowman–Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities. Int. J. Mol. Sci.2024, 25, 5578.
Kim, W.-S.; Gillman, J.D.; Kim, S.; Liu, J.; Janga, M.R.; Stupar, R.M.; Krishnan, H.B. Bowman–Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities. Int. J. Mol. Sci. 2024, 25, 5578.
Kim, W.-S.; Gillman, J.D.; Kim, S.; Liu, J.; Janga, M.R.; Stupar, R.M.; Krishnan, H.B. Bowman–Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities. Int. J. Mol. Sci.2024, 25, 5578.
Kim, W.-S.; Gillman, J.D.; Kim, S.; Liu, J.; Janga, M.R.; Stupar, R.M.; Krishnan, H.B. Bowman–Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities. Int. J. Mol. Sci. 2024, 25, 5578.
Abstract
Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included into animal feed mixtures due to the presence of Kunitz (KTi) and Bowman Birk protease inhibitors (BBi) which reduces animal performance. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBi) but such treatment is energy intensive, adds expense, and negatively impact the quality of seed proteins. As an alternative approach we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays and q15RT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss of function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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