Field Crop 2025, Vol.8, No.2, 72-81 http://cropscipublisher.com/index.php/fc 77 5 Case Study: Successful CRISPR-Based Modification in High-Oleic Soybean and Lessons for Rapeseed 5.1 Background of CRISPR success in soybean The oleic acid content in soybean oil has always been an important indicator, after all, it is related to the quality of the oil. Recently, CRISPR/Cas9 technology has been very useful in this regard. Speaking of which, the FAD2gene is a key role-the enzyme it encodes converts oleic acid into linoleic acid. If the function of this gene is inhibited, the oleic acid content will naturally increase (Wu et al., 2020). However, in actual operation, things are not that simple. Studies have found that editing the FAD2 gene can indeed significantly increase the oleic acid content while reducing the proportion of polyunsaturated fatty acids (Huang et al., 2020; Liu et al., 2022a). But interestingly, there are some differences in the results obtained by different laboratories (Shi et al., 2022). The latest study by Zhou et al. (2023) pointed out that more influencing factors may need to be considered. For example, the genetic background of some soybean varieties may affect the editing effect, which brings some uncertainty to the actual operation. From an application perspective, this technology-modified soybean oil has great potential in the market. Not only is the nutritional value higher, but the industrial processing performance is also better. However, to achieve large-scale application, some technical details may need to be resolved. After all, the promotion of gene-edited crops is always accompanied by various discussions and considerations. 5.2 Key strategies for targeting similar pathways in rapeseed The success story of soybeans has inspired us, and perhaps a similar approach can be used on rapeseed. However, the situation of rapeseed is much more complicated. After all, it is polyploid, and there are several copies of the FAD2 gene (called BnaFAD2). This means that multiple gene sites must be edited at the same time to see a significant effect. In practice, researchers have found that editing two specific sites, BnaFAD2.A5 and C5, has a good effect (Huang et al., 2020). Oleic acid content does increase, but the extent of the increase varies from variety to variety (Liu et al., 2022a). Interestingly, if mutations of different alleles are combined, the effect may be better. Of course, the technical difficulty of this multi-target editing has also increased accordingly. From an application perspective, this gene-edited rapeseed seed is indeed very promising. However, compared with soybeans, gene editing of rapeseed faces more challenges, after all, its genome is more complex. This also explains why related research progress is relatively slow. 5.3 Potential adaptations in protocols to suit rapeseed's genetic profile Editing the rapeseed genome is indeed a technical job. After all, its complex allotetraploid structure is no joke. In order to deal with multiple gene copies, the CRISPR experimental protocol must be specially designed. For example, designing multiple gRNAs for the conserved region of the BnaFAD2 gene is a good way to ensure more comprehensive editing. However, interestingly, studies have found that using a single gRNA can sometimes achieve good results (Zhang et al., 2019). Of course, the multi-gRNA strategy is usually safer. But having a good editing protocol is not enough, and the transformation and regeneration links are also critical (Liu et al., 2022a). After all, in order to make the edited traits stably inherited, every step of the entire process must be controlled well. In actual operation, it will be found that the protocols adopted by different laboratories may be very different. Some prefer a simple and direct single gRNA, while others insist on a multi-gRNA strategy. This is not to say who is right or wrong, the key depends on the specific research goals and variety characteristics. 5.4 Outcome and insights gained from soybean CRISPR modifications CRISPR technology has been used successfully in soybeans, which is indeed of great reference value for rapeseed research. You see, by increasing the oleic acid content and reducing those not-so-good polyunsaturated fatty acids, the quality of the oil will be different immediately (Huang et al., 2020). And the best thing is that this kind of gene editing does not require the introduction of exogenous genes, which not only meets regulatory requirements but is also more acceptable to consumers (Shi et al., 2022). But then again, rapeseed is much more complicated than soybeans. The experience of soybeans tells us that it is important to identify the key genes for fatty acid synthesis (Liu et al., 2022a). Now everyone is focusing on the BnaFAD2 gene, but how to adjust the experimental plan to
RkJQdWJsaXNoZXIy MjQ4ODYzNA==