Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 140 Review Article Open Access Multiplex CRISPR-Cas9 Editing of Yield-related Genes in Rice Mingliang Jin, Danyan Ding Institute of Life Sciences, Jiyang College of Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding email: danyan.ding@jicat.org Rice Genomics and Genetics, 2025, Vol.16, No.3 doi: 10.5376/rgg.2025.16.0013 Received: 05 Apr., 2025 Accepted: 16 May, 2025 Published: 02 Jun., 2025 Copyright © 2025 Jin and Ding, 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: Jin M.L., and Ding D.Y., 2025, Multiplex CRISPR-Cas9 editing of yield-related genes in rice, Rice Genomics and Genetics, 16(3): 140-149 (doi: 10.5376/rgg.2025.16.0013) Abstract Rice yield is a critical determinant of global food security, yet it remains constrained by complex genetic and environmental factors. With the emergence of CRISPR-Cas9 as a powerful genome-editing tool, its application in rice improvement-especially via multiplex gene editing-has gained significant momentum. This study outlines the mechanisms of CRISPR-Cas9 editing in plants, recent advances in multiplex editing strategies, and delivery systems. We focus on key yield-related genes in rice, such as those influencing grain size (e.g., GS3), plant architecture (e.g., DEP1), and stress tolerance (e.g., DRO1), and highlight technological innovations including tRNA-processing systems, base editing, and transgene-free approaches. A case study on the simultaneous editing of GS3, DEP1, and DRO1 demonstrates the feasibility and potential of multiplex CRISPR-Cas9 to enhance multiple yield traits concurrently. Despite technical challenges like off-target effects and regulatory barriers, multiplex CRISPR-Cas9 editing presents a promising frontier in precision rice breeding. Future research integrating advanced CRISPR technologies with precision breeding platforms may accelerate the development of high-yield, climate-resilient rice varieties. Keywords CRISPR-Cas9; Multiplex genome editing; Rice yield; GS3; Precision breeding 1 Introduction Rice yield is very important for global food security. However, there are still many difficulties, such as insufficient genetic diversity, high environmental pressure, and the complexity of yield traits. These problems have been affecting the improvement of rice yield. In recent years, the development of genome editing, especially CRISPR-Cas9 technology, has provided a new and more accurate and efficient way to improve crops (Chen and Zhang, 2024). In the process of rice cultivation, both biotic stress (such as pests and diseases) and abiotic stress (such as drought and high temperature) will affect yield (Lyu, 2024). Moreover, yield traits are usually controlled by multiple genes and quantitative trait loci (QTL), which also makes breeding more difficult. Although traditional breeding has some results, the process is slow and expensive. It takes a long time to get good alleles, and the mutagenesis process also has a certain degree of randomness. In the context of intensified climate change and growing population, we need a fast, accurate and efficient way to breed rice more than ever (Rengasamy et al., 2024; Thiruppathi et al., 2024). CRISPR-Cas9 is a genome editing tool. It can accurately modify genes related to yield and other agronomic traits. Unlike traditional breeding, this technology can modify multiple genes at the same time, which is called "multiple editing". This can breed new rice varieties with high yield, high quality and strong stress resistance more quickly. Researchers have successfully used it to modify key genes such as Gn1a, DEP1, GS3 and IPA1, improving rice grain number, panicle shape and grain shape (Li et al., 2016; Zhou et al., 2018; Zeng et al., 2020). Multiple CRISPR-Cas9 editing can also integrate multiple desirable traits in one generation, while helping us better understand complex gene networks (Xie et al., 2015; Shen et al., 2017). In recent years, multiple CRISPR-Cas9 editing has been increasingly used in improving rice yields, but what actual results have been achieved behind the technology actually requires a systematic review. Therefore, this study focuses on the progress that has been made in this area, especially the technical means and effects of multi-gene editing. There are many studies, some of which are effective but also have problems. We try to sort out several key results and see what they may mean for rice breeding and even food security. Of course, this
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