Legume Genomics and Genetics 2025, Vol.16, No.2, 72-80 http://cropscipublisher.com/index.php/lgg 72 Feature Review Open Access CRISPR/Cas9-Mediated Knockout of Trypsin Inhibitor Genes in Soybean Xingzhu Feng Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding email: xingzhu.feng@hibio.org Legume Genomics and Genetics, 2025 Vol.16, No.2 doi: 10.5376/lgg.2025.16.0008 Received: 30 Jan., 2025 Accepted: 15 Mar., 2025 Published: 05 Apr., 2025 Copyright © 2025 Feng, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Feng X.Z., 2025, CRISPR/Cas9-mediated knockout of trypsin inhibitor genes in soybean, Legume Genomics and Genetics, 16(2): 72-80 (doi: 10.5376/lgg.2025.16.0008) Abstract Trypsin inhibitors (TIs) in soybean are known to have antinutritional effects, reducing protein digestibility and limiting the nutritional value of soy products and animal feeds. To address this long-standing challenge, genome editing tools such as CRISPR/Cas9 have emerged as promising strategies for precisely eliminating undesirable traits such as TIs. This study explores the application of CRISPR/Cas9 to targetedly ablate trypsin inhibitor genes in soybean, specifically those encoding Kunitz and Bowman-Birk inhibitors. We discuss the biological functions and limitations of these inhibitors, outline the mechanisms and recent technical improvements of CRISPR/Cas9, and detail methods for identifying TI gene targets using transcriptomic and proteomic analyses. We also review guide RNA design, translational techniques, and gene editing validation. Functional assessments demonstrated that knockout lines exhibited reduced TI activity, improved protein digestibility, and improved nutritional status, with minimal adverse effects on agronomic traits. A case study demonstrating the successful ablation of the Kunitz trypsin inhibitor gene further demonstrates the utility of this approach. We also explore biosafety concerns, regulatory frameworks, and public perception issues surrounding genome-edited crops. Ultimately, this study highlights the transformative potential of CRISPR/Cas9 for improving the nutritional quality of soybeans and supports future efforts to integrate genome editing into breeding programs to develop high-protein, low-antinutrient varieties. Keywords CRISPR/Cas9; Trypsin Inhibitors; Soybean; Genome Editing; Nutritional Enhancement 1 Introduction Although soybeans are an important source of protein for humans, they are not always so "friendly" to eat. Especially for livestock that require high-protein feed or people with limited protein intake, some "extra components" in soybeans may be a problem. Anti-nutritional factors like trypsin inhibitors (TI) are not fatal in themselves, but they do affect the digestion and absorption efficiency of proteins, thereby reducing their nutritional value. The two more common types of TI-Kunitz type (KTI) and Bauman-Burke type (BBI)-are highly present in soybean seeds. They inhibit the activity of trypsin, an enzyme that is crucial for the normal digestive process in humans and animals. This kind of "inhibition" can lead to proteins not being fully utilized. Over time, it may have a negative impact on the growth and production efficiency of animals. In fact, there were solutions in the past. Heat treatment is one of the traditional methods to remove TI, but its problems are also obvious-it consumes a lot of energy, is costly, and may even "heat" away the nutritional value of the soybeans themselves. Therefore, this approach is not suitable for large-scale, cost-sensitive food and feed industries (Wang et al., 2022). It was not until the emergence of gene-editing tools like CRISPR/Cas9 that things began to change. Rather than "killing" TI by heating, it is better to directly "cut off" them at the genetic level. After key genes like KTI1 and KTI3 were knocked out, the content of inhibitors in soybeans decreased significantly, while the growth and development of the plants themselves were not affected. This approach is not only more accurate but also avoids many limitations of traditional methods (Kim et al., 2024). Even better, the mutation information generated during the gene editing process can also be used to develop molecular markers, accelerating the breeding of new soybean varieties with low TI and high nutrition (Wang et al., 2022).
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