可诱导型CRISPRi系统的构建及其初步应用  

Establishment of an inducible CRISPRi system and its preliminary application

作　　者：陈新玉[1] 庞德剑 欧小利[1] 姜勇[1] 梅柱中[1] 

机构地区：[1]南方医科大学病理生理学教研室广东省功能蛋白质组学重点实验室,广东广州510515

出　　处：《生物技术》2015年第2期128-132,共5页Biotechnology

基　　金：广东省教育厅科技创新项目("天然反义RNA调控TIRAP基因表达的分子机制研究";No.2013KJCX0040)资助~~

摘　　要：[目的]构建可诱导型CRISPRi系统以特异性地抑制靶基因的表达。[方法]采用PCR技术将人ZNF10基因的KRAB结构域编码序列分别克隆至d Cas9蛋白编码基因的5’端(KRAB-d Cas9)与3’端(d Cas9-KRAB)。设计并构建靶向荧光素酶报告基因起始密码子附近序列的4种gRNA表达载体,其表达受到含有Tet O调控元件的H1启动子调控。分析共表达d Cas9蛋白和gRNA对共转染的荧光素酶报告基因表达水平的影响。在此基础上,设计并构建靶向人KLHL21基因转录起始位点附近区域的3种gRNA表达载体,将其与KRAB-d Cas9融合表达载体分别共转染HEK293T细胞,24h后收集细胞并采用荧光实时定量PCR与蛋白质印迹技术分析KLHL21基因表达水平的变化。[结果]CRISPRi系统有效地抑制HEK293T细胞中荧光素酶报告基因与内源性的KLHL21基因的表达。共表达TetR抑制蛋白可阻断CRISPRi对荧光素酶报告基因表达的抑制作用,在细胞培养基中加入1μg/ml盐酸四环素可解除这种抑制作用。[结论]成功构建了可诱导型CRISPRi系统,可有效抑制真核细胞基因的表达。[ Objective] To establish an inducible CRISPR interference （CRISPRi） system for efficiently inhibiting the expression of eu- karyotic genes. [ Methods] The eDNA sequence encoding the KRAB domain of human ZNFIO gene was amplified by PCR. It was then at- tached to the 5＇ -and 3＇ - teminus of dCas9 gene by using overlap extension PCR. These chimeric eDNAs were subsequently subeloned into pcDNA4 plasmind respectively. Four guide RNA （gRNA） plasmids targeting to the adjacent region of the start codon of luciferase eDNA in pGL3 control vector were constructed. The expression of gRNA was under the control of an inducible H1 promoter which contained two Te- tO sites. Their effects on the co - expressed luciferase were analyzed by dual - luciferase reporter assay system. Based on the above results, three gRNA plamsids targeted to the around region of transcription start site of human KLHL21 gene were constructed. They were then co - transfected with KRAB - dCas9 into HEK293T cells. The expression of endogenous KLHL21 was then analyzed by using real - time PCR and western blotting. [ Results] CRISPRi system efficiently suppressed the expression of both the ectopieally expressed lnciferase reporter gene and endogenous KLHL21 gene. Co - expression of TetR blocked the inhibitory effects of the CRISPRi system on the expression of lu- ciferase. And this blockage effect can be released in the presence of 1 μg/ml tetracycline. [ Conclusion] We have successfully constructed an inducible CRISPRi system which can be used to suppress the expression of eukarvotic genes.

关 键 词：CRISPRi 四环素调控表达系统 荧光素酶报告基因 KLHL21基因 

分 类 号：R334[医药卫生—人体生理学]