Design and investigation of CRISPRi tools based on dCasMINI protein
10.16352/j.issn.1001-6325.2024.06.0821
- VernacularTitle:基于dCasMINI蛋白的CRISPRi工具设计及其效果探究
- Author:
Xinwen CHEN
1
;
Jiaxuan CAO
;
Shuquan RAO
Author Information
1. 中国医学科学院血液病医院 (中国医学科学院血液学研究所) 血液与健康全国重点实验室 国家血液系统疾病临床医学研究中心 细胞生态海河实验室,天津 300020;天津医学健康研究院,天津 301600
- Keywords:
CRISPR interference;
dCasMINI;
knock-down;
tetracycline-on(tet-on)
- From:
Basic & Clinical Medicine
2024;44(6):821-827
- CountryChina
- Language:Chinese
-
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.
