Legume Genomics and Genetics 2025, Vol.16, No.2, 72-80 http://cropscipublisher.com/index.php/lgg 74 which also slows down the breeding pace. Ultimately, traditional methods are somewhat lagging behind, which is why there are more highly anticipated new technologies, such as genome editing-directly and precisely "turning off" genes related to TI might be the future direction. 3 CRISPR/Cas9 System 3.1 Mechanism of action: Cas9-mediated DNA double-strand breaks and repair Initially, CRISPR/Cas9 was not discovered for gene editing. In fact, it originated from a natural "defense mechanism" used by bacteria and archaea to resist viral invasion. What truly turned it into a tool was that scientists discovered the "scissors" of this system-the Cas9 protein, as well as the sgRNA (single-guide RNA) that could guide it to its target. The working mode of this combination is actually not complicated: sgRNA will "lead" Cas9 to find the DNA region with a specific PAM sequence. Once the position is matched, Cas9 will cut that DNA and create a double-strand break. Now it all depends on how the cells respond. It may directly "suture" the wound (non-homologous terminal connection, NHEJ), but this repair method is often not very precise and is prone to losing some bases, resulting in the loss of gene function. It is also possible to use templates for repair (homologous directed repair, HDR), in which case precise modifications can be added (Jiang and Doudna, 2017; Zheng et al., 2023). 3.2 Advantages over previous gene editing tools (e.g., TALENs, ZFNs) When it comes to gene editing, in fact, CRISPR/Cas9 is not the first "player". In the early years, there were also ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nuclease). But those who have used them all know that those methods have very high requirements for protein modification. Every time a new target is designed, a new protein has to be reconstructed, which is time-consuming and laborious. CRISPR/Cas9 is much easier in this regard. You only need to change a segment of the sgRNA sequence to make it "cut" different targets. The process is simple and the efficiency is high. This system also supports simultaneous editing of multiple genes, which is particularly convenient for the modification of complex traits. Moreover, it is less sensitive to the background of the DNA sequence or whether it is methylated than previous generations of tools, with fewer restrictions (Arora and Narula, 2017; Manghwar et al., 2019; Janik et al., 2020). 3.3 Recent improvements: multiplex editing, promoter-specific targeting, and off-target minimization In recent years, the application of CRISPR/Cas9 has become more diverse and is no longer merely about "single-point precise strikes". For instance, it is now possible to simultaneously target multiple sites with several Sgrnas at one time to achieve multi-gene editing (Rao et al., 2022). Some people have also attempted to confine the "scissors" function of Cas9 to specific sites or time periods by using tissue-specific or promoter specific expression systems, thereby enhancing operational flexibility (Allemailem et al., 2024). The problem of missing the target was indeed worrying at first: What if the wrong place was cut? However, there have been many improvements in this area now, such as the high-fidelity Cas9 version, the optimized sgRNA design scheme, and even making Cas9 into a ribonucleoprotein complex, and even using exosomes for delivery... All kinds of means are helping to "correct" this problem (Ding et al., 2016; Horodecka and Duchler, 2021). It can be said that these advancements have truly enabled CRISPR/Cas9 to move from laboratory tools to more practical breeding and biotechnology application scenarios. 4 Targeting Trypsin Inhibitor Genes in Soybean 4.1 Identification of candidate TI genes through transcriptomic and proteomic profiling The trypsin inhibitor gene (TI) in soybeans is not easy to recognize at a glance. To know which genes are the "main forces behind the scenes", scientists have to start from both the transcriptome and proteome levels. Through these analyses, the Kunitz (KTI) and Bowman-Birk (BBI) families gradually emerged, and they are basically the main sources of TI activity. For instance, the KTI1 and KTI3 genes (Glyma01g095000 and Glyma08g341500 respectively) are particularly active in seeds, and both expression data and real-time PCR indicate that they are the main "targets". This also makes them the preferred targets for gene editing (Jofuku and Goldberg, 1989; Rosso et al., 2021). The results of proteomics also confirm this point: once these two genes mutate, the content and activity of TI protein will be significantly reduced.
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