人胚胎干细胞多基因同时抑制系统的建立  

Inducible multiplexed CRISPR interference system in human embryonic stem cells

作　　者：朱超南 陈沁雯 辛晨歌 李慧[1] ZHU Chao-nan;CHEN Qin-wen;XIN Chen-ge;LI Hui(Department of Histoembryology,Genetics & Development,Shanghai Jiao Tong University College of Basic Medical Sciences,Shanghai 200025,China)

机构地区：[1]上海交通大学基础医学院组织胚胎学与遗传发育学系

出　　处：《上海交通大学学报（医学版）》2019年第5期478-486,共9页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市自然科学基金(19ZR1428300)~~

摘　　要：目的·在人胚胎干细胞(human embryonic stem cells,hESCs)中构建基于CRISPR 干扰系统的多基因同时抑制系统,作为研究家族基因功能和构建多基因疾病模型的工具。方法·通过Golden Gate 克隆法在hESCs 中建立受强力霉素(doxycycline,Dox)调控的多基因CRISPR 干扰系统。该系统主要由2 个质粒组成:1 个质粒在Dox 调控下融合表达核酸酶失活的CRISPR 相关蛋白9(nucleasedeactivatedCRISPR-associated protein 9,dCas9)和Krüppel 相关盒(Krüppel-associated box,KRAB)抑制结构域(dCas9-KRAB),另1 个质粒可同时表达8 个独立的导向RNA(guide RNA,gRNA)来分别引导dCas9-KRAB 蛋白到基因组特定位点抑制转录起始或延伸。以基因组DNA 为模板进行PCR,确定2 个质粒是否整合至细胞基因组,并通过Western blotting 确定细胞在Dox 诱导下是否可表达dCas9-KRAB 蛋白。结果·使用这种可调控的多基因CRISPR 干扰系统,可在Dox 诱导下在hESCs 中同时成功抑制多个基因的表达;并且这些基因的表达被抑制导致hESCs 形态改变,碱性磷酸酶活性下降,hESCs 表面标志物阶段特异性胚胎表面抗原4(stagespecificembryonic antigen 4,SSEA4)的表达下降,hESCs 发生分化。结论·该CRISPR 干扰系统可以在hESCs 中高效地同时抑制多个基因的表达,是十分有效的多基因研究工具。Objective · To generate a doxycycline (Dox)-inducible multiplexed CRISPR interference (CRISPRi) system for multiple gene inhibition in human embryonic stem cells (hESCs) to explore the function of gene families and model multigene diseases. Methods · A Dox-inducible multiplexed CRISPRi system was developed by Golden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nucleasedeactivated CRISPR-associated protein 9 (dCas9) and Krüppel-associated box (KRAB) transcriptional repressor domain under the control of Dox, the other carrying eight independent guide RNA (gRNA) expression cassettes. PCR was conducted using total genomic DNA as a template to confirm whether these two plasmids were integrated into genome. Western blotting was performed to confirm whether the expression of dCas9-KRAB could be induced by Dox treatment. Results · Using this tunable CRISPRi system, multiple genes were successfully silenced simultaneously in hESCs. The silence of genes and related to hESC self-renewal caused obvious cell differentiation in terms of changed cell morphology, decreased activity of alkaline phosphatase, and reduced expression of stage-specific embryonic antigen 4 (SSEA4), a marker of undifferentiated hESCs. Conclusion · This Dox-inducible multiplexed CRISPRi system can be used for quick and efficient silence of multiple genes in hESCs in a highly controlled manner.

关 键 词：CRISPR干扰系统 可诱导的多基因同时抑制 GoldenGate克隆法 人胚胎干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]