Rice Genomics and Genetics 2025, Vol.16, No.4, 237-244 http://cropscipublisher.com/index.php/rgg 240 3.3 Transformation methods and rice material selection In rice, the most commonly used method for introducing CRISPRi vectors into plants is Agrobacterium-mediated transformation. This method is highly efficient and transgenic can be stably integrated (Butt et al., 2018; Fiaz et al., 2019). The selection of materials is also very crucial. Japonica rice varieties like "Nihon Haru" are widely used because they are easy to transform and regenerate. Selecting healthy and uniformly growing seedlings and then optimizing the tissue culture conditions can further increase the success rate of transformation and also make subsequent analysis more accurate. 4 Applications of CRISPRi in Rice Gene Function Analysis 4.1 Gene silencing and functional validation CRISPRi can precisely target and inhibit the expression of specific genes. Researchers can use it to "silence" genes and then observe the changes in plants to verify the functions of the genes. This method is particularly suitable for rapid gene inhibition and can also be used for high-throughput screening. It is a powerful complement to gene knockout or RNA interference (Bendixen et al., 2023). Because it does not modify the DNA sequence, it can be used more safely and flexibly in functional annotations (Larson et al., 2013). 4.2 Systematic study of gene family functions In rice, members of different gene families sometimes compensate for each other, resulting in knockout mutants not showing obvious phenotypes. Combining tools such as CRISPRi and CAFRI-Rice can simultaneously target multiple members within a gene family, thereby reducing this redundancy (Hong et al., 2020). This makes it easier to identify which genes are primary and which are standby, and also enables a more comprehensive mapping of the role of gene families in the growth and development of rice (Sanson et al., 2018). 4.3 Integration with transcriptomics and metabolomics analyses The process by which CRISPRi inhibits genes is reversible and can also regulate strength, making it highly suitable for combined analysis with transcriptomics and metabolomics. Researchers can first suppress a certain gene using CRISPRi, and then conduct RNA sequencing and metabolite detection to see what downstream changes will be caused (Hong et al., 2020; Bendixen et al., 2023). This multi-omics combined approach can more clearly discover new gene functions and regulatory mechanisms, and also make functional genomics research more precise. 5 Case Studies: CRISPRi Applications in Key Rice Trait Research 5.1 Functional repression studies of flowering time regulation genes CRISPR gene editing has been employed to study genes that control flowering time and plant type, such as IPA1 (ideal plant type 1). When the IPA1 gene mutates, rice will exhibit different tillering characteristics, which indicates that it plays a significant role in flowering and plant morphology regulation. These studies have shown that using CRISPR to inhibit or modify flower-related genes can reveal their multi-faceted effects on developmental and yield traits (Figure 2) (Li et al., 2016; Ricroch et al., 2017; Butt et al., 2018). 5.2 Functional analysis of genes related to grain quality regulation CRISPR/Cas9 is widely used in the research and improvement of rice quality, including appearance, chalkiness and nutritional components. For instance, large-scale mutant libraries of seed-related genes have identified many candidate genes that affect amylose content, protein levels and starch viscosity. Knocking out specific genes, such as Chalk3, can explain the genetic basis of chalkiness in rice and its impact on endosperm structure and composition, which also provides a new target for quality improvement (Fiaz et al., 2019; Zhao et al., 2024). 5.3 CRISPRi functional studies of stress-resistance-related genes CRISPR technology is also used to study genes related to stress resistance such as drought resistance, salt resistance and disease resistance. It can rapidly complete the identification and verification of stress-resistant genes, providing support for the breeding of rice varieties that are more resistant to environmental stress (Ricroch et al., 2017).
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