Rojas-Fernandez Alejandro, Herhaus Lina, Macartney Thomas, Lachaud Christophe, Hay Ronald T, Sapkota Gopal P
Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Sci Rep. 2015 Apr 29;5:9811. doi: 10.1038/srep09811.
CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.
CRISPR/Cas9技术已被用于基因组编辑，以实现体细胞中的基因敲除和敲入。同样，某些内源基因已被荧光蛋白标记。通常，标记蛋白的检测需要高表达以及诸如共聚焦显微镜和流式细胞术等复杂工具。因此，需要一种由内源启动子驱动的简单、灵敏且稳健的转录报告系统来研究转录调控。我们报告了一种基于CRISPR/Cas9的方法，该方法利用转化生长因子β（TGFβ）反应性基因PAI-1，在体细胞中以内源启动子控制下快速整合萤火虫荧光素酶基因。我们的策略采用了一个包含非融合绿色荧光蛋白（GFP）的多顺反子盒，以确保检测转基因传递并快速分离阳性克隆。我们证明萤火虫荧光素酶cDNA可以有效地传递到TGFβ反应性基因PAI-1启动子的下游。使用TGFβ信号的化学和遗传调节剂，我们表明它模拟了内源PAI-1表达的转录调控。我们独特的方法有可能加快在体细胞天然染色质环境中对任何基因转录的研究，从而实现强大的高通量化学和遗传筛选。
