Bowman–Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities

Won Seok Kim, Jason D. Gillman, Sunhyung Kim, Junqi Liu, Madhusudhana R. Janga, Robert M. Stupar, Hari B. Krishnan

Research output: Contribution to journalArticlepeer-review

Abstract

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman–Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts 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 qRT-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.

Original languageEnglish (US)
Article number5578
JournalInternational journal of molecular sciences
Volume25
Issue number11
DOIs
StatePublished - Jun 2024

Bibliographical note

Publisher Copyright:
© 2024 by the authors.

Keywords

  • antinutritional factors
  • Bowman–Birk inhibitor
  • CRISPR-Cas9 enabled mutagenesis
  • Glycine max (L. (Merr))
  • soybean

PubMed: MeSH publication types

  • Journal Article

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