Two-color CRISPR imaging reveals dynamics of HSV-1 replication compartments and virus-host interactions

This data submission contains the raw microscopy images for our manuscript, which describes the following: Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we have developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring ~600 bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split-GFP in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently contact with each other but do not mix, suggesting that RC territory is highly stable. Lastly, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR/Cas9-based imaging tools to study DNA viruses in living cells.