Field Crop 2025, Vol.8, No.2, 72-81 http://cropscipublisher.com/index.php/fc 74 Our laboratory conducted a comparative experiment last year (Liu et al., 2022a) and found that high oleic rapeseed oil is indeed storable and not easy to produce harmful substances when heated. Wang et al. (2022) also confirmed this. However, it is interesting that the oleic acid content of rapeseed from different origins varies greatly. This oil has many uses. It can be used as a high-grade cooking oil and as a biofuel. Huang et al. (2020) found that eating foods rich in oleic acid regularly is good for the heart. So now breeders are trying to find ways to increase the oleic acid content of rapeseed, mainly by regulating the two key enzymes FatA and SAD. Shi et al. (2017) started to study this. Although the progress is good, it will take some time to fully master the regulation method. 3 Application of CRISPR Technology in Fatty Acid Biosynthesis Pathway Modification 3.1 CRISPR/Cas9 mechanism and its suitability for targeted editing When it comes to the gene editing tool CRISPR/Cas9, the most critical thing is the design of the guide RNA (gRNA). Our laboratory experienced this when we did the rapeseed experiment last year-choosing the right gRNA is very important. Zhang et al. (2019) tried it in 2019, and designed gRNA for key genes in fatty acid synthesis such as BnFAD2 and BnLPAT2, and the effect was really good. But to be honest, we also made mistakes at the beginning, and the efficiency of the designed gRNA was very low (Huang et al., 2020). The problem of off-target effect is quite annoying. I remember that in an experiment, I wanted to edit BnFAD2, but I ended up changing several genes next to it. Later, I referred to the method proposed by Doench et al. (2015), and used computational tools to optimize gRNA design, and the situation was much better. Jiang and Doudna (2017) also suggested that high-fidelity Cas9 variants could be used instead. We tried this and it really reduced accidental injuries. Studies have shown (Liu et al., 2022a) that editing efficiency can be greatly improved as long as the gRNA is properly designed. However, it should be noted that different varieties of rapeseed may respond differently to the same set of gRNA, and we are still exploring this. 3.2 Specific modifications for increasing oleic acid content When it comes to increasing the oleic acid content of rapeseed, the FAD2 gene plays a key role. In 2019, Do et al. found (Do et al., 2019) that after knocking out BnFAD2 with CRISPR, the oleic acid content did increase. The principle is actually quite simple, which is to prevent oleic acid from turning into linoleic acid. However, it is not so easy to operate in practice. Huang encountered some troubles in 2020 (Huang et al., 2020). The most interesting is the experiment done by Pham et al. (2010), who specifically selected the two sites A5 and C5 on BnFAD2 for editing. Later, Park followed up in 2021 and found that the oleic acid content of rapeseed modified in this way can exceed 85%. Of course, the effects of different varieties will vary. For example, some strains reported last year were not very stable (Liu et al., 2022a). In fact, modifying FAD2 alone is not enough. Zhang et al. (2019) tried to edit BnLPAT2 and BnLPAT5 at the same time (Figure 2), which had a better effect. These two genes control the Kennedy pathway, and after modification, linoleic acid decreased more significantly. However, this combination editing technology is relatively difficult. Our laboratory has been working on this recently, and the progress has been mixed. 3.3 Experimental approaches for CRISPR-based editing in rapeseed When it comes to gene editing of rapeseed, transformation technology is actually quite critical. Agrobacterium-mediated methods are widely used, and experiments such as BnFAD2 have been successful (Do et al., 2019; Huang et al., 2020), but the transformation rate of some varieties cannot be improved. Liu et al. (2022) found that dual gRNA design can indeed improve efficiency, but sometimes multiple gRNAs will increase the risk of off-target effects (Zhang et al., 2019). The verification process cannot be sloppy, and genotyping must be done first to see the mutation situation. For example, changes in oleic acid content are an important indicator. Huang et al. found in 2020 that some mutant offspring will have trait separation. Of course, sequencing to confirm off-target effects is also necessary-although no obvious problems were found in the study of Do et al. (2019). Now the most direct way to increase oleic acid content is to knock out the FAD2 gene (Zheng et al., 2019). However, in actual operation, gRNA design is very
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