Design and investigation of CRISPRi tools based on dCasMINI protein

Abstract

Objective To explore the design of CRISPR interference (CRISPRi) tools based on the deactivated CasMINI (dCasMINI) protein and to evaluate their transcriptional inhibition effects. Methods The tetracycline-on (tet-on) system, flow cytometry, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to evaluate the transcriptional inhibition effects of dCasMINI system in mammalian cells at three level-plasmid genes, exogenous genomic loci, and endogenous genomic loci. Additionally, six dCasMINI-CRISPRi tools (dCasMINI, dCasMINI-ZIM3 KRAB, dCasMINI-KRAB-MeCP2, dCasMINI-ZNF324 KRAB, dCasMINI-3x KRAB, and dCasMINI-Com-KRAB-MECP2) were designed and compared for their transcriptional inhibition effects along with single guide RNA (sgRNA) at different positions. Results dCasMINI, dCasMINI-ZIM3 KRAB,dCasMINI-KRAB-MeCP2, dCasMINI-ZNF324 KRAB, dCasMINI-3x KRAB, and dCasMINI-Com-KRAB-MeCP2 exhibited varying degrees of transcriptional inhibition on plasmids genes and exogenous genomic genes (P＜0.05). Additionally, dCasMINI-ZIM3 KRAB, dCasMINI-KRAB-MeCP2, dCasMINI-ZNF324 KRAB, and dCasMINI-Com-KRAB-MeCP2 demonstrated different levels of transcriptional inhibition on endogenous genes (P＜0.05). Different positions of sgRNAs showed distinct transcriptional inhibition effects (P＜0.05). Conclusions The CasMINI system can be adapted into various CRISPRi tools for gene knockdown studies, with potential applications in various scenarios such as epigenetic gene editing in primary cells, in vivo screening, and clinical therapy in the future.

Keywords

• crispr interference|dcasmini|knock-down|tetracycline-on (tet-on)