Wu Mengyao, Liu Senquan, Gao Yongxing, Bai Hao, Machairaki Vasiliki, Li Gang, Chen Tong, Cheng Linzhao
Division of Hematology, Huashan Hospital of Fudan University, Shanghai, China; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Stem Cell Res. 2018 May;29:6-14. doi: 10.1016/j.scr.2018.03.003. Epub 2018 Mar 10.
Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.
在人类诱导多能干细胞（iPSC）中进行精确的基因组编辑，显著提高了我们将人类iPSC用于疾病建模、药物测试与筛选以及人类细胞生物学研究的能力。在本研究中，我们试图实现CD55基因的条件性表达，以便探究其功能。我们使用了两种具有独特基因型的人类iPSC系，并构建了一种可诱导的Cas9基因表达系统，该系统整合于人类基因组的AAVS1安全位点。使用配对的引导RNA，我们观察到包括CD55和ETV6基因在内的几个基因的编码区出现了预期缺失的高效敲除。这种配对引导RNA方法使我们能够有效地鉴定出具有预期缺失的纯合iPSC克隆。一旦鉴定并表征了缺乏CD55表达的iPSC克隆，我们就能够使用相同的强力霉素系统在未分化和分化的iPSC中诱导来自猪尾巴病毒载体的CD55转基因表达。这种基因敲除与可诱导转基因互补的单细胞系足以实现CD55基因的条件性表达。这里描述的方法广泛适用于其他基因，以探究它们的功能。
