Recent advancements in genetic engineering technology have led to the development of CRISPR interference (CRISPRi) as a precise tool for regulating gene expression. When CRISPR/dCas9 is fused with transcriptional repressors, the system can robustly silence endogenous gene expression. The CRISPR/Cas9 tool is a promising alternative in organisms (e.g., Bombyx mori) that do not respond to traditional gene suppression techniques, such as RNA interference (RNAi). However, transcriptional repressors remain poorly categorized in multiple cell types and species, leading to difficulties in optimizing performance and efficiency. Here, we tested CRISPRi usability and efficiency in Bombyx mori cells (BmE). We fused dCas9 to five transcriptional repressors including KRAB, Hairy, SID, SRDX, and ERD. All five constructs were efficient in BmE cells. In a proof-of-concept experiment, we showed that CRISPRi acting on BmSoxE (a gene involved in cell proliferation) could generate similar phenotypes as RNAi gene suppression. Moreover, CRISPRi has fewer off-target effects. Through co-transfection of BmE cells with sgRNAs, we also demonstrated that dCas9 could simultaneously repress the expression of multiple genes. Furthermore, we identified sgRNA distance from transcriptional start site (TSS) and the dCas9: sgRNA ratio as the two limiting factors of CRISPRi efficiency. Our results demonstrated that CRISPR/dCas9 is a viable and rapid alternative for functional investigations of the B. mori genome and perhaps other Lepidoptera insects.

中文翻译：

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使用 CRISPRi 系统抑制 BmE 细胞内源基因的转录。


基因工程技术的最新进展导致 CRISPR 干扰 (CRISPRi) 的发展，作为调节基因表达的精确工具。当 CRISPR/dCas9 与转录抑制子融合时，该系统可以强有力地沉默内源基因表达。对于对传统基因抑制技术（如 RNA 干扰 (RNAi)）没有反应的生物体（例如家蚕）来说，CRISPR/Cas9 工具是一种很有前景的替代方案。然而，转录抑制因子在多种细胞类型和物种中的分类仍然很差，导致优化性能和效率的困难。在这里，我们测试了 CRISPRi 在家蚕细胞 (BmE) 中的可用性和效率。我们将 dCas9 与五种转录抑制子融合，包括 KRAB、Hairy、SID、SRDX 和 ERD。所有五个构建体在 BmE 细胞中均有效。在概念验证实验中，我们表明 CRISPRi 作用于 BmSoxE（一种参与细胞增殖的基因）可以产生与 RNAi 基因抑制相似的表型。此外，CRISPRi 的脱靶效应较少。通过与 sgRNA 共转染 BmE 细胞，我们还证明 dCas9 可以同时抑制多个基因的表达。此外，我们确定 sgRNA 与转录起始位点 (TSS) 的距离和 dCas9: sgRNA 比率是 CRISPRi 效率的两个限制因素。我们的结果表明，CRISPR/dCas9 是一种可行且快速的替代方案，可用于家蚕基因组以及其他鳞翅目昆虫的功能研究。