Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the recently emerged virus responsible for the COVID-19 pandemic. Clinical presentation can range from asymptomatic disease and mild respiratory tract infection to severe disease with lung injury, multiorgan failure, and death. SARS-CoV-2 is the third animal coronavirus to emerge in humans in the 21st century, and coronaviruses appear to possess a unique ability to cross borders between species and infect a wide range of organisms. This is somewhat surprising as, except for the requirement of host cell receptors, cell–pathogen interactions are usually species-specific. Insights into these host–virus interactions will provide a deeper understanding of the process of SARS-CoV-2 infection and provide a means for the design and development of antiviral agents. In this study, we describe a complex analysis of SARS-CoV-2 infection using a genome-wide CRISPR-Cas9 knock-out system in HeLa cells overexpressing entry receptor angiotensin-converting enzyme 2 (ACE2). This platform allows for the identification of factors required for viral replication. This study was designed to include a high number of replicates (48 replicates; 16 biological repeats with 3 technical replicates each) to prevent data instability, remove sources of bias, and allow multifactorial bioinformatic analyses in order to study the resulting interaction network. The results obtained provide an interesting insight into the replication mechanisms of SARS-CoV-2.
Highlights
SARS-CoV-2 (isolate 026V-03883; kindly granted by Christian Drosten, Charité—Universitätsmedizin Berlin, Germany by the European Virus Archive—Global (EVAg)) and mock-infected samples were generated by infecting confluent monolayers of Vero E6 cells at 400 TCID50 /mL
To identify cellular factors required for SARS-CoV-2 infection, we used a human
100,000 unique gRNAs in total), and 1000 non-targeting gRNAs to perform a genomewide CRISPR-Cas9 screen in HeLa cells expressing the angiotensin-converting enzyme 2 (ACE2) entry receptor (HeLaACE2 ), which is highly permissive to the SARS-CoV-2 infection resulting in CPE development and cell death [22]
Summary
The employment of semi-random or broad-spectrum gRNA libraries allows for the inactivation of several genes in a cell population and high-throughput analysis of cellular pathways, involved in the response to viral infection This approach has already been used for the identification of the vast array of host factors involved in the pathogenesis of many viruses: coronaviruses [9,10,11,12,13], ZIKV [14], HIV-1 [15], or Influenza A virus (IAV) [16]. The developed analysis pipeline allows for an unbiased assessment of the cellular factors required for the productive SARS-CoV-2 infection and may be used in the future for other pathogens
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