Fig. 1: CLCC1 emerged as a top positive regulator of HSV-1 nuclear egress in a genome-wide CRISPR-Cas9 screen.

a Schematic of the nuclear egress assay. HeLa cells are infected with HSV-1 encoding tdTomato, permeabilized, and stained with the capsid-specific primary antibody and Alexa-488-conjugated secondary antibody. Infected cells with nuclear egress have capsids in the cytoplasm are stained, whereas cells without nuclear egress will lack staining due to capsid retention in the nucleus. b Flow cytometry readouts from the validation of the nuclear egress assay. HeLa cells were infected with HSV-1 F GS3217-d34 (dUL34 HSV-1) or HSV-1 F GS3217 (WT HSV-1). Uninfected HeLa cells served as a control. The y-axis (red, tdTomato) indicates HSV-1 infection, and the x-axis (green, Alexa-488) indicates the presence of capsids. Each image is representative of three replicates. c Immunofluorescence images from validation of the nuclear egress assay. Experimental conditions are the same as in (b). Scale bar = 10 µm. Each image is representative of three biological replicates. d Schematic of the genome-wide CRISPR screen. HeLa-Cas9 cells were transduced with Gattinara library lentivirus, cells were subsequently infected with HSV-1 F GS3217, stained and sorted by flow cytometry. e Representative flow data from the genome-wide CRISPR screen. Graph is from one of the six sorting rounds, where two independently generated Gattinara library HeLa cells were each infected with HSV-1 on three independent occasions. f Volcano plot of the genome-wide CRISPR screen. Each dot represents a specific gene. The x-axis shows the average log₂ fold change (FC) of the specific gene. The y-axis shows the significance score plotted as -log(p-value). The dotted line at y = 3 is the threshold for p-value = 0.001 [−log₁₀(p-value) = 3). The dashed line at y = 1.3 is the threshold for p-value = 0.05 [−log₁₀(p-value) = 1.3). Red: top hit, CLCC1. Green: genes known to contribute to HSV-1 nuclear egress. High-confidence hit, EMD, is labeled. Light gray: non-site targeting sgRNAs. Dark gray: intergenic site targeting sgRNAs. The statistics were calculated using two-side paired t-text without correction for multiple comparisons and converted to the −log₁₀(p-value). Source data are provided in Source Data 1–4 and Supplementary Data 1. Figure 1a: Schematic of the nuclear egress assay. Created in BioRender. Dai, B. (2025) https://BioRender.com/s1ge6ad. Figure 1d: Schematic of the genome-wide CRISPR screen. Created in BioRender. Dai, B. (2025) https://BioRender.com/99sttvr.