Legume Genomics and Genetics 2025, Vol.16, No.2, 72-80 http://cropscipublisher.com/index.php/lgg 75 4.2 Design of guide RNAs for precise editing of KTI and BBI gene clusters Editing the TI gene is not something that can be accomplished simply by "cutting" it. For the CRISPR/Cas9 system to function effectively, it must first have a suitable gRNA (guide RNA) to lead the way. For the KTI and BBI gene clusters, the design of grnas is usually based on their respective sequence-specific regions and expression data. Current research typically targets the open reading frames of KTI1 and KTI3 with gRNA, allowing Cas9 to create small insertions or deletions in these regions, thereby "inactivating" these genes. This operation is not done all at once but can hit multiple targets simultaneously, greatly enhancing efficiency. As for BBI, the strategy is basically similar, and loss-of-function mutants can also be obtained, ultimately achieving the goal of reducing the activity of inhibitory proteins. 4.3 Use of tissue culture and transformation systems for delivery of CRISPR constructs Loading CRISPR tools into soybean cells is not as simple as just "tapping and importing". The method commonly used by researchers is Agrobacterium-mediated, and the objects of operation are generally the cotyledon segments or embryonic tissues of soybeans. Agrobacterium can help "send in" the CRISPR system, allowing it to be stably expressed within cells. Afterwards, through tissue culture, complete plants can be cultivated from these edited cells. Then, screening and genotype analysis are still necessary to confirm which individuals actually carry the desired mutations. For those who need to switch to breeding projects, it is necessary to identify the double-homozygous mutants without exogenous DNA again. This process, although cumbersome, has been proven effective in the editing experiments of KTI and BBI, and has greatly accelerated the development of low-TI soybean varieties. 5 Outcomes of Gene Knockout: Functional and Agronomic Evaluation 5.1 Reduction in trypsin inhibitor activity confirmed by biochemical assays Has the gene been cut in the right place? Just imagining is not enough; one still needs to look at the data from biochemical analysis. For editing systems like CRISPR/Cas9, whether the target protein is finally handled or not usually depends on the detection of protein activity or abundance to confirm. The FAD2 gene is a typical example. After being pruned, its fatty acid composition changed significantly, which is basically evidence that it was "knocked out" (Zhang et al., 2023). Although the current activity data for TI is not yet sufficient, it does not mean that no one has made similar attempts. Methods like RT-qPCR and protein quantification are still relatively common approaches in confirming the success of gene knockout and the decline in protein levels. 5.2 Unintended effects on plant growth, seed viability, or yield Can plants still grow normally after one gene is cut off? This is the problem that many people are most worried about. To figure this out, researchers conducted numerous "physical examinations" on these gene-edited soybean and rice strains. Most of the results are still quite optimistic. For instance, several soybean varieties that had FAD2 knocked out grew quite normally, with no change in yield and even taller plant types. Some even demonstrated better resistance to lodging. On the rice side, after knocking out certain target genes, no significant differences were observed in pollen viability and yield (Kim et al., 2019). But then again, the absence of obvious external changes doesn't mean nothing has happened. Some potential metabolic or physiological fluctuations may require more sophisticated assessment methods to be detected (Bouche and Bouchez, 2001). 5.3 Enhanced protein digestibility and improved nutritional profile of edited soybean lines At present, many studies focus on "visible" properties such as oleic acid content and fatty acid profile, but logically speaking, changing the target can also work. Anti-nutritional factors such as trypsin inhibitors (TI), if reduced through gene knockout, theoretically would enhance the digestibility of protein, which would be beneficial to the nutritional value of soybeans. Previous successes in oilseed traits, such as the promotion of high oleic acid soybean varieties, have already demonstrated that gene editing can indeed lead to quality improvement. On this basis, editing for TI is expected to enhance the protein utilization efficiency of soybeans, thereby providing a new breakthrough for breeding improvement.
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