应用CRISPR/Cas9系统构建Rev-erbβ基因敲除的HEK293细胞系

Construction of Rev-erbβ gene knockout HEK293 cell line with CRISPR/Cas9 system

目的利用成簇的、规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9)基因组编辑技术,构建Rev-erbβ基因敲除的HEK293细胞系。方法通过单向导RNA(sgRNA)介导Cas9蛋白对目的基因靶位点DNA进行特异性的切割,然后经DNA同源重组单向导RNA或非同源末端连接方式进行修复,以实现对Rev-erbβ基因进行敲入、敲除修饰操作的目的。首先,针对Rev-erbβ基因设计4个sgRNA,经筛选选择活性较高的sgRNA1及sgRNA2用于构建p CMV-h Cas9-U6-Rev-erbβsgRNA1&sgRNA2串联载体。然后将p CMV-h Cas9-U6-Rev-erbβsgRNA1&sgRNA2和p Ad-E1/hRev-erbβdonor质粒载体共转染至HEK293细胞,通过药物筛选、克隆化及序列测序获得整合有外源供体基因片段的一条链,另一条链为片段缺失的Rev-erbβ基因完全敲除的HEK293(Rev-erbβ-/-)细胞系。最后通过用Western blot法和实时定量PCR对敲除Rev-erbβHEK293细胞系(C3-6)进行检测。结果敲除Rev-erbβ基因的HEK293细胞系中均未检测到Rev-erbβmRNA和蛋白质的表达。结论利用CRISPR/Cas9技术,成功构建了基因定点修饰和敲除的Rev-erbβ-/-HEK293细胞系,为Rev-erbβ的功能和作用机制研究提供有效工具。

Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease（CRISPR/Cas9） gene editing technology.Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA（sgRNA）-mediated Cas9 cutting of target DNA,and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair.Firstly,four sgRNAs were designed for Rev-erbβ gene.The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct p CMV-h Cas9-U6-Reverbβ sgRNA1 and p CMV-h Cas9-U6-Rev-erbβ sgRNA2.Then,p CMV-h Cas9-U6-Rev-erbβ sgRNA1,p CMV-h Cas9-U6-Reverbβ sgRNA2 and p Ad5-E1/hRev-erbβ donor plasmid vectors were co-transfected into HEK293 cells.Through drug screening,cloning and sequencing,the Rev-erbβ gene-knockout HEK293（Rev-erbβ^-/-） cell lines were obtained with one chain integrated with exogenous gene fragment and the other chain for deletion mutants.Finally,the HEK293（Rev-erbβ^-/-）cell lines（C3-6） was detected with real-time quantitative PCR and Western blotting.Results Expression of Rev-erbβ mRNA and protein was undetectable in HEK293 Rev-erbβ^-/-cell line.Conclusion Using CRISPR/Cas9 technology,the HEK293Rev-erbβ^-/-cell line has been successfully constructed,which would provide an effective tool for the study on the function of Rev-erbβ.