Gene Expression
Nebulas
Gene Expression NebulasSummary: Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 in Vero-E6 cells, identifying known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex, the alarmin HMGB1, and the transcription factors Smad3 and Smad4. Small molecule inhibitors of these pathways inhibited SARS-CoV-2-infection in both monkey and human cells, demonstrating the conserved role of these genetic hits across species. We also revealed that HMGB1 is a novel regulator of ACE2 expression and critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
Overall Design: RNAseq, ChIPseq (H3K4me3, H3K27ac) and ATACseq of Vero-E6 cell lines in control sgRNA and HMGB1 KO sgRNA conditions.
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| Growth Protocol: | Vero-E6 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), and 1% Penicillin/Streptomycin. |
| Treatment Protocol: | - |
| Extract Protocol: | Cells were infected with SARS-CoV-2 at a MOI of 1 for 24 hours then RNAs were extracted using Direct-zol RNA MiniPrep Kit and submitted to the Yale Center for Genome Analysis for library preparation. |
| Library Construction Protocol: | Libraries were prepared using TruSeq stranded mRNA library prep kit (Illumina) |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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