Legume Genomics and Genetics 2025, Vol.16, No.2, 72-80 http://cropscipublisher.com/index.php/lgg 76 6 Case Study 6.1 Experimental setup: cultivar selection, editing strategy, and transformation technique When many people do soybean gene editing, their first reaction is to use Williams 82 (abbreviated as WM82). The reason is not complicated: The genomic information of this variety is relatively complete, it is widely used, and the data is also complete. So for this experiment, the WM82 was also chosen for the trial operation. The target genes are KTI1 (Gm01g095000) and KTI3 (Gm08g341500)-they are basically the "main force" of TI activity in soybean seeds (Figure 2). The research team designed guide RNA (gRNA) to specifically target the open reading frames of these two genes, inducing small fragment deletions or insertions, thereby rendering them "ineffective". As for how to deliver the CRISPR system into soybeans, the method remains the old one: Agrobacterium transformation + tissue culture. By sending the construct into the explant and then through tissue regeneration, the edited plant can be obtained (Wang et al., 2023). 6.2 Validation of mutations: sequencing results, inheritance, and expression analysis It needs to be verified whether the cut is on the right spot after it is done. Researchers first confirmed through sequencing that frameshift mutations had indeed occurred in KTI1 and KTI3, with small insertions or deletions causing the loss of gene function. Protein detection further clarified the issue-the edited KTI1 protein was truncated and had lost its ability to inhibit trypsin. Interestingly, the study also identified a T1 generation strain (#5-26), which simultaneously carries homozygous mutations of kti1 and kti3, and does not carry the exogenous Cas9 gene. This indicates that mutations can be stably inherited and the expected traits can be retained without the use of genetically modified components. In addition, expression analysis also supported this result: functional KTI1 and KTI3 proteins were no longer detectable in this type of edited plants. 6.3 Results and implications: reduction in anti-nutritional activity and enhanced food/feed value Has TI's level dropped or not? The result is crystal clear. The KTI content and trypsin inhibitory activity of the double mutant were significantly lower than those of the wild-type WM82 and the commercially available varieties. If ranked by the strength of inhibitory activity, it would roughly be: KTI1/3 double mutant < kti1 single mutant ≤ known low TI germplasm < WM82 < commercial control. The most crucial point is that although KTI1 and KTI3 were cut off, this did not affect the plant's own growth, seed development or maturation. In other words, what should grow will still grow, and what should be tied will still be tied. This gives people a lot more confidence in the practical application of these editing strains: not only is the protein digestibility higher and the nutritional value better, but also the cost of heat treatment during processing is saved. Moreover, these achievements can also provide a basis for the subsequent development of low-Ti molecular markers, indirectly accelerating the breeding process. 7 Biosafety, Regulatory, and Ethical Considerations 7.1 Regulatory landscape for genome-edited crops across major jurisdictions For genome-edited crops, such as soybeans modified with CRISPR/Cas9, the regulation of each country cannot be generalized. In the United States, the regulatory mechanism may sound complex, but generally speaking, the Department of Agriculture (USDA), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA) each have their own areas of responsibility. They will make judgments based on technical features and product uses instead of labeling "genetically modified" right from the start. In recent years, the United States has also been considering simplifying procedures and integrating biosafety and biosecurity for unified management (Beeckman and Rudelsheim, 2020; Cao, 2021; Li et al., 2021). Looking at the EU side from the opposite perspective, its attitude is rather strict. At present, they treat all new genomic technology products, including CRISPR, as genetically modified organisms (GMO). However, the situation is also changing. The EU is already discussing whether to distinguish between traditional genetically modified and precisely mutagenic products (Ongu et al., 2023). China also has specific regulations. The Biosecurity Law, which was introduced in 2020, provides a governance framework. However, there are still many unanswered questions regarding how to implement it and how to respond to the rapid iteration of technology (Han et al., 2020). As for some developing countries, such as certain regions in Africa, although they have established a biosecurity framework, practical
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