Abstract
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.
Highlights
The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution
The method relies on ribosomal DNA genes in Saccharomyces cerevisiae, which visually stand out from the surrounding chromatin and act as landmarks for the localization of specific genes that have been deliberately inserted for analysis
In this work we establish a method for inserting genes between ribosomal DNA (rDNA) loci in S. cerevisiae, which thereby enriches protein–DNA complexes and enables the ex vivo visualization of macromolecules transcribing, modifying or binding DNA sequences in a native-chromatin environment
Summary
The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. 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. CLEM may not be useful when the target feature looks similar to its surroundings, as is the case for specific genes within the nucleus Technologies such as DNA paint[3], the sequence-specific binding of labeled proteins[4], and invasive methods[5,6] that explore, e.g., electron-dense particles inserted in the cell do not allow for the localization and identification of specific genes. We incorporated multiple copies of two RNA Pol IIItranscribed genes: the nucleolar locus-native RDN5 (5 S rRNA) and the locus-foreign HSX1 (arginine tRNA)[8]
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