Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 146 7.2 Integration with precision breeding platforms Multiple CRISPR editing technology is now gradually being combined with precision breeding platforms. Researchers have developed more efficient systems and simpler vector designs, such as the SSTU (single transcription unit) method, which allows multiple guide RNAs to work simultaneously. This allows for faster combination, transformation, and screening of different trait combinations (Wang et al., 2018). These technologies can help breeders bring together multiple good genes, and also analyze how each gene affects each other, ultimately enabling faster breeding of new rice varieties with high yield, good quality, and strong stress resistance (Shen et al., 2017; Mishra et al., 2018). 7.3 Policy, biosafety, and global adoption Whether the technology itself can be promoted sometimes depends not only on the effect, but also on whether it can pass the policy test. Especially for tools like multiple CRISPR, once the words "genetic modification" are involved, some countries will be particularly sensitive. Because of this, many research teams now prefer to go the "non-GMO" and "unlabeled" route. This is not because the technology itself is not good, but to bypass the psychological defense line of the public and regulatory authorities (Ren et al., 2019; Pan and Qi, 2023). But even if you don't carry foreign DNA, there is no guarantee that all countries will recognize it. The standards in various places are varied, some are loose, and some are strict, making it difficult to implement in practice. If you really want to make this technology widely implemented, it may not be realistic to rely on one country, one policy. At least there must be a unified general framework across countries. Moreover, supervision is only one step, and it is more difficult to get the public to buy in. Problems such as off-target effects and ecological impacts are not small. Whether it is out of scientific prudence or to increase transparency, follow-up research cannot be stopped. Only when these risk points are clearly explained and managed can CRISPR truly go far and stand firm (Biswas et al., 2020). 8 Concluding Remarks Multiple CRISPR-Cas9 editing techniques have been shown to simultaneously and precisely modify multiple key yield-related genes, such as Gn1a, DEP1, GS3, and IPA1. Editing these genes can effectively increase the number of rice grains, the structure of the panicle, and the size of the grains. With this technique, researchers can quickly obtain plants with one, two, or even three mutations in the target genes. These mutations combined can also produce a stronger yield-enhancing effect, thereby significantly increasing the single panicle yield of excellent rice varieties. If genes related to yield and stress resistance are edited at the same time, higher-yielding and more resistant rice lines can also be bred. These new results simplify the entire breeding process, making it easier to combine good genes in one variety and to analyze how different genes work together. However, despite the rapid technological progress, some problems still exist. Off-target effects and unexpected mutations are not common, but they do occur. This requires extra caution in the design and use of guide RNAs. In addition, multi-gene editing may lead to conflicts or interactions between traits, and the results may not be easy to predict, so careful screening and verification in the field are required. In addition, the construction process of the editing vector is relatively complicated, and the editing efficiency of different gene sites is different. These technical problems have not been completely solved. At the same time, the regulatory rules of different countries are not unified, and clear and unified biosafety standards are still being formulated, which have affected the promotion of gene-edited rice. Overall, multiple CRISPR-Cas9 editing technology has great development potential in accelerating rice breeding and increasing grain production. As long as the technology is continuously optimized and can be better combined with the precision breeding platform, coupled with the continuous improvement of non-GMO methods, the application value of this technology will become greater and greater. In the future, if we can further solve the technical, biological and policy problems, multiple gene editing can play an important role in rice breeding more safely and efficiently.
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