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.本次提交的数据包含对应研究手稿所用的原始显微成像图像，该手稿阐述了以下内容：用于单病毒追踪的实时成像工具可实现病毒复制与病毒-宿主相互作用的空间分辨定量测量。然而，在活宿主细胞中同时高效标记亲本病毒与子代病毒仍是一项挑战。本研究开发了一种基于CRISPR标签系统（CRISPR-Tag system）的新型策略，用于检测宿主细胞内的1型单纯疱疹病毒（herpes simplex virus 1, HSV-1）DNA。我们构建了携带约600 bp CRISPR标签序列的重组1型单纯疱疹病毒，该序列可被dCas9-荧光蛋白（FP）融合蛋白有效识别。CRISPR辅助单病毒基因组追踪（CRISPR-assisted single viral genome tracking, CASVIT）技术使我们能够在单细胞水平上解析病毒复制的时空信息。结合SunTag与串联分裂GFP（tandem split-GFP）在扩增荧光信号方面的优势，我们构建了dSaCas9-tdTomato10x与dSpCas9-GFP14x，以实现高效的双色CASVIT检测。实时双色成像结果显示，复制区室（replication compartments, RCs）频繁相互接触但并未发生融合，这表明复制区室的区域边界具有高度稳定性。最后，双色CASVIT技术可同时追踪病毒DNA与宿主染色质，研究发现病毒复制早期阶段宿主细胞内的端粒与着丝粒DNA会发生显著丢失。综上，本研究建立了一套基于CRISPR/Cas9的成像工具开发框架，用于研究活细胞内的DNA病毒。