Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 142 Figure 1 Schematic overview of strategies for the generation and isolation of transgene-free edited plants (Adapted from Son and Park, 2022) Image caption: (A-C) Strategies for the isolation of transgene-free edited plants. (D-F) Strategies for the generation of edited plants without the stable integration of a transgene. Several CRISPR/Cas9 systems allow gene editing without requiring the stable integration of a transgene. (G) Transgene-free genome editing based on haploid induction. (H) The in planta genome editing based on in planta particle bombardment (iPB) method (Adapted from Son and Park, 2022) 3.2 Plant architecture genes The appearance of rice is not only determined by leaves and height, but also by the shape of the panicle and the number of tillers, which are closely related to yield. The DEP1 gene is related to the structure of the panicle. If it is edited with CRISPR, rice will usually grow more compact and upright panicles, and the plant height will be reduced (Wang et al., 2017). Such plants are more resistant to lodging and the harvest is often more stable. However, not all plant type modifications can go smoothly. Genes like IPA1 have no problem regulating plant type, but their mutation effects are related to the target of OsmiR156, which sometimes increase the number of tillers and sometimes reduce them (Li et al., 2016). It depends on the specific situation. There is also OsFWL4, which is a member of the FW2.2 class of genes. It has a "suppressive" effect on tillering and yield. Studies have found that as long as this gene is knocked out, the number of tillers and yield will often increase (Gao et al., 2020). 3.3 Stress tolerance genes linked to yield Unexpected events happen all the time, and rice is not always grown in an ideal environment. When saline-alkali land, drought, and adverse conditions come, the yield can easily decline. Therefore, whether the stress resistance is strong or not is also the key. CRISPR-Cas9 was used to knock out a gene called OsRR22. It was found that the salt tolerance of rice increased, but other agronomic traits were basically unaffected (Zhang et al., 2019). This kind of "improvement without side effects" editing result is actually rare, which is a bright spot. There is another idea, which is to target certain miRNAs and indirectly regulate functional factors such as OsGRF4 through them, which can also improve stress resistance and increase yield by the way (Yadav et al., 2023; Lin et al., 2021). 4 Advances in Multiplex CRISPR-Cas9 Editing Systems in Rice 4.1 tRNA-processing and Csy4-based systems If you want to move multiple genes in a rice plant at the same time, you really can't do it without some skills. What researchers use more now is the tRNA processing system that plants have. It is not a new invention, but a "borrowed" set of mechanisms that plants already have - the specific method is to connect multiple tRNA-gRNA
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