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

作　　者：陈信文 曹佳璇 饶书权 CHEN Xinwen;CAO Jiaxuan;RAO Shuquan(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,CAMS&PUMC,Tianjin 300020;Tianjin Institutes of Health Science,Tianjin 301600,China)

机构地区：[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 [2]天津医学健康研究院,天津301600

出　　处：《基础医学与临床》2024年第6期821-827,共7页Basic and Clinical Medicine

基　　金：天津市自然科学基金(21JCQNJC01220)。

摘　　要：目的探索基于去活化CasMINI蛋白(dCasMINI)的CRISPR干扰(CRISPRi)工具设计及其转录抑制效果评估。方法采用四环素诱导基因开启系统(tet-on),流式细胞测量术和实时荧光定量反转录聚合酶链反应从质粒基因、基因组外源性基因位点和内源性基因位点三个层次评估dCasMINI系统在哺乳动物细胞中的转录抑制效果,并进一步设计和比较6种dCasMINI-CRISPRi工具(dCasMINI、dCasMINI-ZIM3 KRAB、dCasMINI-KRAB-MeCP2、dCasMINI-ZNF324 KRAB、dCasMINI-3x KRAB和dCasMINI-Com-KRAB-MeCP2)和不同位置单链引导RNA(single guide RNA,sgRNA)的转录抑制效果。结果dCasMINI、dCasMINI-ZIM3 KRAB、dCasMINI-KRAB-MeCP2、dCasMINI-ZNF324 KRAB、dCasMINI-3x KRAB和dCasMINI-Com-KRAB-MeCP2能够不同程度地抑制质粒基因、基因组外源性基因的转录(P<0.05);dCasMINI-ZIM3 KRAB、dCasMINI-KRAB-MeCP2、dCasMINI-ZNF324 KRAB和dCasMINI-Com-KRAB-MeCP2能够不同程度地抑制内源性基因的转录(P<0.05);不同结合位点的sgRNA具有不同的转录抑制效果(P<0.05)。结论CasMINI系统能够改造成多种CRISPRi工具进行基因敲降研究,未来有望应用在多个场景,如原代细胞表观基因编辑、体内筛选和临床治疗等。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) 

分 类 号：R34[医药卫生—基础医学]