Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy

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. Manger, Ermel, and Frangakis report the use of CRISPR/Cas9 to stably insert multiple copies of a particular gene-of-interest near the 35S rRNA gene, to allow direct visualization of gene clusters with electron microscopy. They achieve more than 7-fold enrichment of associated Pol III complexes within the field of view, demonstrating the utility of the approach.

Automated summary

Manger, Ermel, and Frangakis demonstrate the use of CRISPR/Cas9 technology to stably insert multiple copies of a gene-of-interest near the 35S rRNA gene, enabling direct visualization of gene clusters with electron microscopy. They achieve a more than 7-fold enrichment of associated Pol III complexes within the field of view, showcasing the effectiveness of this approach.