Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 141 technology is far from perfect. There are also many troubles such as off-target effects and regulatory restrictions, so they must also be discussed. As for the future, whether this technology can become an important tool for sustainable improvement, it still needs to be judged in combination with existing challenges and development trends. 2 Overview of CRISPR-Cas9 Technology in Plants 2.1 Basic mechanism of CRISPR-Cas9 editing This gene editing tool was actually first discovered from the immune mechanism of bacteria. After all, the principle of CRISPR-Cas9 is not complicated, but people who come into contact with it for the first time may still find it a bit mysterious. In plants, its operation process is as follows: researchers first design a molecule called "single guide RNA" (sgRNA), and then this RNA will accurately bring the Cas9 protein to the target position of the DNA. Then, Cas9 cuts the DNA at that point, and the two chains are broken. After that, the plant itself will start the repair mechanism and try to fill the break, which often results in gene mutations. This method is not only efficient, but also not difficult to use, so it is now very popular in breeding and plant research (Belhaj et al., 2015; Bortesi and Fischer, 2015; Liu et al., 2017). Of course, although the principle is simple, some technical accumulation is still required for actual operation. 2.2 Development of multiplex genome editing strategies Not every edit changes just one gene. Now, researchers often want to "do multiple things at once," or multiple edits. The key to this approach is to design multiple sgRNAs and then have them enter plant cells together with Cas9. In this way, several important traits, such as disease resistance, stress resistance, or yield, can be improved at the same time. In the past, such edits were not easy to do, mainly due to the design of vectors and RNA. Now it is different. These technologies have matured a lot and are easier to operate (Ma et al., 2016; Wada et al., 2020). Interestingly, this way of editing multiple genes at once can not only quickly superimpose ideal traits, but also allow us to more intuitively see how these genes work with each other. Sometimes, the obvious effect can be seen in one generation of plants, which really saves a lot of trouble in breeding new varieties. 2.3 Regulatory and delivery considerations There are several methods for delivering CRISPR-Cas9-related components into plant cells, such as Agrobacterium-mediated delivery, gene guns, and protoplast transfection. Recently, some people have tried to use ribonucleoprotein complexes or viral vectors for "DNA-free" delivery, which does not require the foreign DNA to remain in the plant and may reduce regulatory issues related to transgenics (Figure 1) (Arora and Narula, 2017; Ma et al., 2020; Son and Park, 2022). Different countries and regions have different regulations on the management of CRISPR technology. In some places, whether it is transgenic or not will be distinguished based on whether it contains foreign DNA. In order to make CRISPR-Cas9 widely used in crop breeding, not only efficient delivery methods are required, but also clear regulatory rules are needed (Gan and Ling, 2022; Liu et al., 2022; Sahoo et al., 2023). 3 Yield-Related Genes Targeted by CRISPR-Cas9 in Rice 3.1 Grain size and weight genes CRISPR-Cas9 technology has been used to edit several key genes that affect grain size and weight. Among them, GS3 is an important gene that controls grain size and is a negative regulator. When this gene mutates, the grains become larger. Another important gene is Gn1a, which encodes a cytokinin oxidase/dehydrogenase. By editing Gn1a, cytokinins in rice panicle tissue can be increased, thereby increasing the number of grains and total yield (Bhavya et al., 2024). In addition, mutations in some genes in the cytochrome P450 family can also make grains larger and increase the number of cells, further promoting yield increases (Usman et al., 2020). Other studies have found that in the OsGRF4 gene, if the target site of miRNA is destroyed, the grains can also become larger.
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