The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.

转录或 DNA-蛋白质结合事件的直接研究需要以分子分辨率对单个基因进行成像。电子显微镜 (EM) 可以显示基因组的局部细节。然而，特定单个基因的直接可视化和分析目前是不可行的，因为它们不能明确地定位在细胞核的拥挤、无地标环境中。在这里，我们提出了一种基因簇的基因组插入方法，这些基因簇可以与它们在 EM 中的相关蛋白质复合物一起定位和成像。该方法使用 CRISPR/Cas9 技术在 35S rRNA 基因附近加入几个感兴趣的基因，这是核仁内经常出现的、易于识别的基因组位点，可用作显微照片中的标志。作为原理证明，我们证明了位点原生基因 RDN5 和位点外源基因 HSX1 的掺入。这导致与视野内基因相关的 RNA 聚合酶 III (Pol III) 复合物的富集超过 7 倍，从而显着提高了分析产量。因此，该方法允许插入和直接可视化基因簇以进行一系列分析，例如细胞或外部因素改变后基因活性的变化。