摘要/Abstract
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（nucleasedeactivated CRISPR-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（stage-specific embryonic 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。
关键词: CRISPR干扰系统, 可诱导的多基因同时抑制, Golden Gate克隆法, 人胚胎干细胞
Abstract:
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 developedGolden 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) 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 of dCas9-KRAB could be inducedDox 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 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.
Key words: CRISPR interference system, inducible multiple gene knockdown, Golden Gate assembly, human embryonic stem cells (hESCs)
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