Rice Genomics and Genetics 2025, Vol.16, No.4, 237-244 http://cropscipublisher.com/index.php/rgg 237 Research Insight Open Access CRISPR Interference (CRISPRi) for Functional Genomics in Rice Xingzhu Feng Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding email: xingzhu.feng@hibio.org Rice Genomics and Genetics, 2025, Vol.16, No.4 doi: 10.5376/rgg.2025.16.0020 Received: 28 Jun., 2025 Accepted: 12 Aug., 2025 Published: 28 Aug., 2025 Copyright © 2025 Feng, 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: Feng X.Z., 2025, CRISPR interference (CRISPRi) for functional genomics in rice, Rice Genomics and Genetics, 16(4): 237-244 (doi: 10.5376/rgg.2025.16.0020) Abstract Functional genomics is an important approach to analyzing the gene functions and complex biological processes of rice. CRISPR interference (CRISPRi) technology has become an efficient, reversible and highly specific gene regulatory tool by using Cas9 (dCas9), which lacks cutting activity, to target and bind to DNA with single-guide RNA (sgRNA), thereby inhibiting gene transcription without causing double-strand breaks. This study systematically introduces the principle and composition of the CRISPRi system, including the dCas9 variant, the design strategy of sgRNA and its transcriptional inhibition mechanism. It reviews the latest progress of CRISPRi in the construction and optimization of rice platforms, and focuses on discussing its application in gene silencing, systematic research of gene families, and combined transcriptome and metabolome analysis. Meanwhile, through typical cases such as flowering time regulation, grain quality improvement and stress resistance enhancement, the application value of CRISPRi in the research of important traits of rice was demonstrated. This study also analyzed the advantages of this technology over the traditional CRISPR-Cas9, as well as the current technical bottlenecks (such as off-target effects, differences in silencing efficiency) and possible directions for improvement. This study aims to comprehensively summarize the research strategies of rice functional genomics based on CRISPRi, identify key challenges, and propose future development directions to accelerate molecular breeding and precise trait improvement of rice. Keywords CRISPRi; Rice; Functional genomics; Gene silencing; Molecular breeding 1 Introduction Rice (Oryza sativa L.) is the staple food for more than half of the world's population. Its genetic improvement is of great significance to global food security. Functional genomics is a systematic approach to studying the functions and interactions of genes, which can identify genes that control important agronomic traits, such as yield, stress resistance and rice quality. Traditional breeding and early genetic tools have driven the improvement of rice, but due to the complexity of the rice genome and the current need for more precise and higher-throughput gene function analysis, more powerful methods are required. Functional genomics can analyze the gene networks behind key traits, helping to identify candidate genes for targeted breeding more quickly. The emergence of genome editing technology, especially the CRISPR system, has made the gene modification of rice more precise and efficient, and enabled the simultaneous regulation of multiple genes. This has greatly accelerated the improvement in terms of output, quality and stress resistance (Miao et al., 2013; Zegeye et al., 2022). CRISPR interference (CRISPRi) is an improvement of the CRISPR/Cas9 system. It uses catalytically inactivated Cas9 (dCas9) that fuses with transcriptional repressors. Under the guidance of sgRNA, dCas9 binds to the promoter of the target gene, blocking transcription without cutting DNA. This method can achieve reversible and tunable simultaneous inhibition of multiple genes, so it is very useful in research that requires gene knockdown rather than knockout. Compared with RNA interference and traditional genome editing, CRISPRi has higher specificity, lower off-target rate, and can also target non-coding regulatory regions (Fiaz et al., 2019; Zhang et al., 2025). This study will review the principles and latest progress of CRISPRi in rice functional genomics, with a focus on its application in analyzing gene functions and regulatory networks. It will also explore its potential in
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