Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 143 units into a string and put them into the same transcript. Next, the processing mechanism in the plant can automatically cut out these guide RNAs and complete the precise positioning and cutting of multiple genes at one time. In other words, it is much easier to do it one by one, and the efficiency is very high, even close to 100% at high times (Xie et al., 2015). This trick can be used not only on rice, but also on other species, because this processing mechanism itself is more "universal". Of course, in addition to the tRNA system, some people have tried to use Csy4. This method relies on bacterial RNA enzymes to cut RNA arrays, and the principle is not complicated. But in the final analysis, the tRNA method is used more in rice editing, and the reason is very direct - it has a simpler structure, runs more stably, and has a lower probability of error. 4.2 CRISPR-Cas variants and base editing In addition to the most common Cas9 enzyme, scientists have also modified other CRISPR enzymes, such as Cpf1 (also called Cas12a), for multiple gene editing in rice. These new systems can express multiple crRNAs under the same promoter, which makes vector construction simpler and can edit more different gene sites (Wang et al., 2018). In addition, there are some special Cas9 variants, such as Cas9-VQR, which can recognize some sequences different from conventional PAM, which also expands the scope of editing (Hu et al., 2017). There is also a technology called "base editing", which is a fusion of Cas9 and cytidine deaminase, which can directly change one base to another without causing DNA double-strand breaks. This method can achieve more precise base mutations in rice and other plants (Shimatani et al., 2017). 4.3 Transgene-free and marker-free editing Genetically modified organisms always sound worrying, especially in places with particularly strict regulations, where even trace amounts of foreign DNA can cause controversy. To solve this problem, researchers have simply bypassed the traditional genetic modification route. Now, they have developed some editing methods that do not involve foreign DNA, such as using RNP complexes or using systems with very short expression times. The advantage of this is that the genes that should be edited are modified, but the foreign substances will not remain in the rice genome for a long time (Arora and Narula, 2017). Of course, it is not enough to just be "DNA-free". In order to really put these materials into use, the problem of screening must be solved. In recent years, high-throughput screening processes have been put online, which can quickly find plants that do not carry genetically modified markers but have multiple desirable traits (Patel-Tupper et al., 2023). Another point is also worth mentioning - the marker-free system. Although it sounds less conspicuous, it is particularly important for subsequent commercialization and regulatory approval. After all, who doesn't want their varieties to look a little "cleaner"? 5 Case Study: Multiplex Editing of GS3, DEP1, andDRO1inRice 5.1 Study overview and experimental design Multiple CRISPR-Cas9 technology can edit multiple yield-related genes in rice at the same time. In a typical study, researchers chose the rice variety "Zhonghua 11" as the test subject. They focused on editing GS3 (controlling grain size), DEP1 (controlling panicle type) and other important genes related to yield. They used the CRISPR/Cas9 system to mutate these target genes and tested the editing effects in the first generation (T0) of transgenic rice. The results showed that this method had a high editing success rate, with an editing rate of 57.5% for GS3 and 67.5% for DEP1, indicating that this multi-gene editing method is effective in improving complex traits (Li et al., 2016). 5.2 Phenotypic and agronomic outcomes Some changes can actually be seen at first glance. For example, the grains of plants edited with GS3 are significantly larger (Figure 2); for another example, the ears of mutants of DEP1 become more compact, facing upward, and even the height of the plants is shorter. These are not unexpected, but expected performance. More importantly, these traits can still remain stable in the next generation (T2) and have not disappeared due to intergenerational replacement. Of course, in addition to these "on-target" results, the researchers also observed some other phenotypes. Some plants have semi-dwarf stalks, while others have obvious long awns. These
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