Legume Genomics and Genetics 2025, Vol.16, No.2, 72-80 http://cropscipublisher.com/index.php/lgg 73 This study will focus on the research progress of CRISPR/Cas9 in reducing TI content in soybeans. Besides the basic background of TI and the current editing strategies, it will also explore the potential of related technologies in commercial breeding programs, as well as the development of molecular markers and other related contents. 2 Trypsin Inhibitors in Soybean: Biological Role and Limitations 2.1 Classification and function of kunitz and Bowman-Birk trypsin inhibitors in plant defense There is actually not just one type of trypsin inhibitor in soybeans. There are mainly two types: KTI (Kunitz type) and BBI (Bauman-Burke type) (Figure 1). KTI mainly targets trypsin, while BBI is more "omnipotent" and also has an inhibitory effect on chymotrypsin. These inhibitors are not only present in seeds but can also be found in multiple parts such as leaves, stems and roots. Almost the entire plant is equipped with this "defense system". Some studies have conducted experiments and found that after overexpressing the genes of KTI or BBI, insects like the corn borer would "keep their distance" from these plants-the number of leaves they ate was significantly reduced (Birdwell et al., 2025). This also indirectly indicates that they are indeed useful for plants in resisting insects. However, when it comes to nematodes as an enemy, these inhibitors are not as effective (Sultana et al., 2023). So, although it has a defensive function, it is not applicable to all pests and diseases. Figure 1 Two configurations of soybean trypsin inhibitors (Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI)) (Adopted from Luo et al., 2025) 2.2 Impact on protein digestibility and animal feed efficiency By the way, these "protective factors" are beneficial to plants, but not good news for animals and humans. TI (trypsin inhibitor) adheres to digestive enzymes such as trypsin and chymotrypsin, disabling them. The result is a reduction in protein absorption. Especially non-ruminants, such as pigs, chickens, and humans, are more affected. If the TI content in soy products is high, the protein digestibility may even drop to around 66%. For whey protein, the content is even higher, reaching 75% (Takacs et al., 2022; Xu et al., 2022). This gap has already been able to affect the weight gain and feed conversion efficiency of animals. Another study has pointed out that a high-Ti diet can cause the pancreas of animals to become larger and its function may also be affected (Gu et al., 2023). So, from a nutritional perspective, the content of TI does indeed need to be controlled. 2.3 Challenges posed by high trypsin inhibitor content in soybean breeding and processing The problem is not merely poor absorption after taking it. The high TI content has also caused a lot of trouble for breeding and subsequent processing. Although heat treatment can remove TI, it comes at a considerable cost-not only does it consume a lot of electricity and have a high cost, but it also destroys certain amino acids in soybeans (Luo et al., 2025). This makes it a bit difficult for processing enterprises that originally intended to produce high-quality protein products. What's more troublesome is that the TI content varies quite a lot among different soybean varieties. This genetic difference makes it particularly complicated for breeders to select varieties with low TI. Moreover, there is still a lack of molecular markers that can quickly screen for low-TI traits at present,
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