RESUMO
SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. IN BRIEFSARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. HIGHLIGHTS- Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types
RESUMO
Population scale sweeps of viral pathogens, such as SARS-CoV-2, that incorporate large numbers of asymptomatic or mild symptom patients present unique challenges for public health agencies trying to manage both travel and local spread. Physical distancing is the current major strategy to suppress spread of the disease, but with enormous socio-economic costs. However, modelling and studies in isolated jurisdictions suggest that active population surveillance through systematic molecular diagnostics, combined with contact tracing and focused quarantining can significantly suppress disease spread1-3 and has significantly impacted disease transmission rates, the number of infected people, and prevented saturation of the healthcare system4-7. However, reliable systems allowing for parallel testing of 10-100,000s of patients in larger urban environments have not yet been employed. Here we describe "COVID-19 screening using Systematic Parallel Analysis of RNA coupled to Sequencing" (C19-SPAR-Seq), a scalable, multiplexed, readily automated next generation sequencing (NGS) platform8 that is capable of analyzing tens of thousands of COVID-19 patient samples in a single instrument run. To address the strict requirements in clinical diagnostics for control of assay parameters and output, we employed a control-based Precision-Recall and predictive Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of over 600 patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads and a sensitivity of > 95% on high viral loads associated with disease onset and peak transmissibility. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.
