Voets Olaf, Tielen Frans, Elstak Edo, Benschop Julian, Grimbergen Max, Stallen Jan, Janssen Richard, van Marle Andre, Essrich Christian
Galapagos BV, CL Leiden, The Netherlands.
PLoS One. 2017 Aug 11;12(8):e0182974. doi: 10.1371/journal.pone.0182974. eCollection 2017.
Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.
使用人原代细胞的表型分析是药物发现中靶点发现和验证的极有价值的工具。在这些分析中对这类靶点进行表达敲低（KD），能够研究它们在疾病过程模型中的作用。因此，在表型分析中需要高效快速的蛋白质KD模式。CRISPR/Cas9系统已被证明是在永生化细胞系中实现基因失活的一种通用且高效的手段。在此，我们描述了腺病毒（AdV）CRISPR/Cas9载体在两种人原代细胞类型（正常人肺成纤维细胞和人支气管上皮细胞）中实现高效基因失活的应用。在转化生长因子-β诱导的成纤维细胞向肌成纤维细胞转变分析（FMT）和上皮向间充质转变分析（EMT）中研究了基因失活的效果，这两种分析依赖SMAD3且反映了在纤维化中观察到的致病机制。AdV Cas9与靶向SMAD3的向导RNA（gRNA）共转导（co-TD），通过插入/缺失（indel）形成判断，导致快速高效的基因组编辑，同时SMAD3蛋白表达和核转位显著降低。这导致了SMAD3抑制下游的表型变化，包括α平滑肌肌动蛋白和纤连蛋白1表达大幅降低，它们分别是FMT和EMT 的标志物。单独的Cas9和gRNA AdV共转导与单个“一体化”Cas9/gRNA AdV转导（TD）的直接比较显示，两种方法实现了相似水平的indel形成。这些数据表明，AdV CRISPR/Cas9是在人原代表型分析中进行蛋白质KD的一种有用且高效的工具。与现有工具相比，AdV CRISPR/Cas9的使用可能具有显著优势，并且应该会增加靶点发现和验证的机会。
