Transcriptional repression of endogenous genes in BmE cells using CRISPRi system.

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.