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As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2., Graphical Abstract, Highlights • CRISPR-Cas13a can quantitatively detect SARS-CoV-2 RNA without pre-amplification • Combining crRNAs targeting multiple regions of the viral RNA enhances sensitivity • Cas13a can accurately and rapidly quantify SARS-CoV-2 RNA in patient samples • A mobile phone-based device allows for portable and sensitive readout of the assay, Fozouni et al. devise a way to use CRISPR-Cas13a to detect and quantify SARS-CoV-2 RNA from patient samples without the need for a pre-amplification step. They then show how the assay’s signal can be efficiently detected with a portable, mobile phone-based device.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies., Graphical Abstract, Highlights • Genome-wide CRISPR screens for SARS-CoV-2, HCoV-229E, and HCoV-OC43 host factors • Screens correctly identified divergent entry factors for the three coronaviruses • Cholesterol and phosphatidylinositol pathways are shared host dependency factors • Pharmacological inhibition of host factors reduces coronavirus replication, To identify host factors required for the infection with SARS-CoV-2 and the common cold coronaviruses OC43 and 229E, Wang et al. conduct genome-wide CRISPR knockout screens. In addition to virus-specific entry factors, they uncover shared host pathways, including cholesterol homeostasis and phosphatidylinositol kinases, required for the infection with all three viruses and demonstrate that pharmacological inhibition of these pathways exhibits pan-coronavirus antiviral activity.