The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.

中文翻译：

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用于 MS2 标记活干细胞中单个 mRNA 的 CRISPR/Cas9 平台

MS2 系统是直接在活细胞中研究单个分子转录动力学的强大工具。过去，在特定基因位点插入 RNA 标记盒一直是一个主要障碍。在这里，我们提出了一种基于 CRISPR/Cas9 的方法，在任何编码基因的 3' 非翻译区的开头插入一个带有选择标记的 MS2 盒。我们通过在小鼠胚胎干细胞中标记干细胞转录因子 Esrrb 的 RNA 来证明我们的方法的适用性。我们使用定量荧光显微镜确定 Esrrb 基因座处新生转录物的数量和表达基因的细胞比例。我们发现，在向外胚层样细胞分化后，Esrrb 的表达在越来越多的细胞中以二元方式下调。