Journal: Molecular Systems Biology
Article Title: Essentiality and dynamic expression of the human tRNA pool during viral infection
doi: 10.1038/s44320-025-00181-7
Figure Lengend Snippet: ( A ) A schematic representation of the experimental setup and distributions of the measured GFP levels of uninfected HFF cells (upper panel), HCMV-infected HFF cells with non-targeting sgRNA (middle panel), and CRISPR-targeted and HCMV-infected HFF cells (lower panel). The percentage of cells in each of the GFP levels, GFP1-4, is marked in the middle and lower panels. ( B ) A volcano plot for targeted gene hits from tRNA-CRISPR screen in HCMV infection. The x-axis shows the Z-score of log2 FC between lowly infected cells (GFP2) and highly infected cells (GFP4). The y-axis shows the –log10 FDR as calculated from MAGeCK. The genes are marked according to the sub-libraries. Significance is determined by FDR < 0.05. All values are calculated for three biological repeats. ( C ) A heat map showing the (−log10) p -value of the hypergeometric test, which tests the enrichment of each sub-library in one of the following groups: significantly enriched in GFP2 cells (log2FC > 0, FDR < 0.05) or significantly depleted in GFP2 cells (log2FC < 0, FDR < 0.05). ( D ) The number of viral genomes estimated by the relative number of UL44 normalized to the B2M human gene, calculated by qPCR, in each individual CRISPR knockout. The dots in each bar depict three or four biological repeats. P -values represent statistical significance of differences between each group and the non-targeting control, as evaluated using Welch’s t-test with Holm correction for multiple comparisons. ( E , F ) Expression level (RPM) determined from tRNA-sequencing of ( E ) iMet-CAT-1-1 gene and ( F ) His-GTG-1-1 gene in cells targeted by non-targeting sgRNA (NT) or by sgRNAs corresponding to the tested tRNA. The dots in each bar depict three biological repeats. ( G ) A heat map describes the z-score log2FC between the different lowly infected cell populations (GFP1, GFP2, GFP3) and the highly infected cells (GFP4) for the significant gene hits. Genes found as significant hits in at least one of the comparisons are presented here. Non-significant hits are marked in gray squares. The genes are colored and marked according to their sub-library, corresponding to the colors and marker shapes described in the legend of Fig. 6B. The dendrogram depicts the similarity between the comparisons based on the enrichment gene hits pattern.
Article Snippet: CRISPR sgRNA design tool of Benchling , https://benchling.com , .
Techniques: Infection, CRISPR, Knock-Out, Control, Expressing, Sequencing, Marker
