基础医学与临床2024，Vol.44Issue(6)：821-827,7.DOI:10.16352/j.issn.1001-6325.2024.06.0821
基于dCasMINI蛋白的CRISPRi工具设计及其效果探究
Design and investigation of CRISPRi tools based on dCasMINI protein
摘要
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.关键词
CRISPR干扰/dCasMINI/敲降/四环素诱导基因开启系统(tet-on)Key words
CRISPR interference/dCasMINI/knock-down/tetracycline-on(tet-on)分类
医药卫生引用本文复制引用
陈信文,曹佳璇,饶书权..基于dCasMINI蛋白的CRISPRi工具设计及其效果探究[J].基础医学与临床,2024,44(6):821-827,7.基金项目
天津市自然科学基金(21JCQNJC01220) （21JCQNJC01220）
