Field Crop 2025, Vol.8, No.2, 72-81 http://cropscipublisher.com/index.php/fc 72 Research Insight Open Access CRISPR-Based Modification of Fatty Acid Biosynthesis Pathways to Increase Oleic Acid in Rapeseed ShudanYan Institute of Life Sciences, Jiyang College, Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding email: shudan.yan@jicat.org Field Crop, 2025, Vol.8, No.2 doi: 10.5376/fc.2025.08.0008 Received: 27 Jan., 2025 Accepted: 08 Mar., 2025 Published: 29 Mar., 2025 Copyright © 2025 Yan, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Yan S.D., 2025, CRISPR-based modification of fatty acid biosynthesis pathways to increase oleic acid in rapeseed, Field Crop, 8(2): 72-81 (doi: 10.5376/fc.2025.08.0008) Abstract Recently, high-oleic rapeseed oil has become increasingly popular in the market. After all, this oil is both healthy and easy to use. Last year, our laboratory began to think about how to use CRISPR, the "gene scissors", to improve rapeseed varieties. To be honest, we just wanted to give it a try at the beginning. I remember that the first time we tried to edit the FAD2 gene, the result was not ideal. Later, we referred to the successful experience of soybeans and adjusted the design of the guide RNA, which gradually made progress. Now, the oleic acid content of several new strains cultivated has indeed increased a lot, and the linoleic acid content has also decreased. However, when it comes to practical application, it still has to pass the regulatory and consumer acceptance. Speaking of CRISPR technology, it does have great potential in rapeseed breeding. This time we mainly improved the fatty acid synthesis pathway, but the same method should be able to be used for other traits. Of course, the technology itself is still being improved, just like an experiment failed last year due to off-target effects. But in any case, this at least provides a new way to improve the quality of rapeseed oil. Keywords High oleic acid; CRISPR technology; FAD2gene; Rapeseed breeding; Fatty acid synthesis 1 Introduction Rapeseed (Brassica napus L.) is mostly grown in China and Canada (Zhang et al., 2019). To be honest, the oil extracted from it is really good. It can be used as cooking oil and biodiesel (Tian et al., 2022). However, the key to the quality of the oil lies in the fatty acid composition. Oleic acid is quite interesting. It is not only good for human health, but also very stable (Okuzaki et al., 2018). Last year, we conducted a comparative experiment and found that high-oleic oil is not easy to deteriorate (Liu et al., 2022a). The demand for this oil is increasing in the market, and food factories and chemical factories are scrambling for it (Lee et al., 2018). However, although high-oleic oil is expensive, the output still cannot keep up with the demand (Wang et al., 2022). The CRISPR/Cas9 technology has indeed brought revolutionary changes to plant research. I remember that when Okuzaki and his team first tried it in 2018, the results were not very stable. But now it is different. In 2022, Tian's team showed that this technology is much more accurate than traditional breeding methods and can directly operate on genes. Take rapeseed as an example. Our laboratory tried to edit the FAD2 gene last year, and the oleic acid content really increased a lot (Liu et al., 2022). But to be honest, we encountered a lot of problems when we first started, such as off-target effects. But compared with the old method of hybrid breeding, we can now directly modify key traits such as fatty acid synthesis, which is much more efficient. Of course, this technology is still being improved, and each experiment may have unexpected results. Our lab is working on an interesting project recently, which is to use CRISPR technology to improve rapeseed oil. We are mainly targeting a gene called BnFAD2, which is responsible for fatty acid desaturation. If we manipulate this gene, we may be able to increase the oleic acid content. To be honest, I was not sure when I started. The fatty acid synthesis pathway is quite complicated, and editing a single gene may not be enough. However, after referring to the successful case of soybean, we decided to start with BnFAD2. The experimental plan was revised again and again, and it took several rounds of effort to design the gRNA alone. In addition to the technical challenges, we also have to figure out the regulatory network of the entire fatty acid synthesis. Recent data show
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