Rice Genomics and Genetics 2025, Vol.16, No.4, 237-244 http://cropscipublisher.com/index.php/rgg 238 accelerating rice genetic improvement and the future prospects of integrating CRISPRi into the breeding process. This study aims to provide an expandable, high-precision and low-risk regulatory platform for the research of rice gene functions, promote the development of functional genomics, and offer theoretical and technical support for rice breeding. 2 Components and Mechanism of the CRISPRi System 2.1 dCas9 and its mutated characteristics (loss of nuclease activity) The CRISPRi system uses an inactivated Cas9 protein called dCas9. It has undergone a mutation on Cas9, which has deprived it of the cutting function of endonuclease. Unlike normal Cas9, dCas9 can find and bind to target DNA without causing double-strand breaks. So it doesn't modify the DNA sequence but reduces gene expression by blocking transcription. It is precisely because it does not cut DNA that this method can achieve controllable and reversible gene suppression (Ma, 2024). 2.2 Design principles of guide RNA (sgRNA) and target site selection CRISPRi requires the use of single guide RNA (sgRNA) to deliver dCas9 to a specific DNA location. Whether sgRNA is well designed or not directly affects its specificity and efficiency. The PAM sequence and the 3' end seed sequence of sgRNA are important in identifying and binding to target sites (Larson et al., 2013). If there is an incomplete match in the seed region, off-target situations may occur. Therefore, special attention should be paid when designing and validating Sgrnas (Rohatgi et al., 2024). In rice research, computational tools and phylogenetic analysis can be combined to select more suitable target sites and also reduce the interference of gene family redundancy (Hong et al., 2020). 2.3 Molecular mechanism of transcriptional blockage and gene expression regulation When the dCas9-sgRNA complex binds to the DNA target, it physically blocks the advancement of RNA polymerase, thereby preventing the initiation or extension of transcription (Larson et al., 2013; Vercauteren et al., 2024). This method does not modify the DNA sequence, but it can effectively and tunably inhibit gene expression. Because it is modular, it is very suitable for high-throughput research on gene functions and regulatory elements, and is very useful for functional genomics studies of rice and other organisms. 3 Construction and Optimization of the Rice CRISPRi System 3.1 Vector construction and promoter selection In rice, the CRISPRi vector typically needs to simultaneously express catalytically inactivated Cas9 (dCas9) and one-way guide RNA (sgRNA). The common approach is to create a binary carrier and assemble these two components together. The selection of the promoter is very important. dCas9 often employs potent constitutive promoters, such as the rice ubiquitin promoter (OsUbi), which ensures its stable expression in various tissues. sgRNA typically uses RNA polymerase III promoters, such as OsU3 or OsU6, because they can efficiently transcrib small Rnas and the starting nucleotides can be set as needed to achieve the best effect of sgRNA. Studies have shown that such promoters can enhance the editing and regulation efficiency of rice (Figure 1) (Butt et al., 2018; Fiaz et al., 2019; Zegeye et al., 2022). 3.2 Efficient sgRNA expression and screening To achieve efficient expression of sgRNA, one can optimize the sgRNA scaffold and select an appropriate promoter. When designing, it is necessary to ensure high targeting activity as much as possible while minimizing off-target activities. This can predict the specificity and efficiency of sgRNA using computational tools (Zegeye et al., 2022). When screening for effective Sgrnas, computer prediction is usually conducted first, followed by experimental verification, such as using rapid detection platforms or observing phenotypic changes to determine the inhibitory effect. There are now some more convenient detection methods, such as instrumentless detection based on CRISPR/SpRY, which can identify CRISPRi events in rice more quickly (Su et al., 2024).
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