Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection (preprint)

SARS-CoV-2 can cause a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of the host factors mediating viral infection or restriction is critical to elucidate SARS-CoV-2 host-pathogen interactions and the progression of COVID-19. To this end, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. These screens uncovered proviral and antiviral host factors across highly interconnected host pathways, including components implicated in clathrin transport, inflammatory signaling, cell cycle regulation, and transcriptional and epigenetic regulation. Mucins, a family of high-molecular weight glycoproteins and the main constituent of mucus, are central components of a prominent viral restriction pathway that we identified. We demonstrate that multiple membrane-anchored mucins are critical inhibitors of SARS-CoV-2 entry and are upregulated in response to viral infection. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and suggests interactions between SARS-CoV-2 and airway mucins of COVID-19 patients as a host defense mechanism.

Non-uniform UV-C dose across N95 facepieces can cause 2.9-log variation in SARS-CoV-2 inactivation (preprint)

During public health crises like the COVID-19 pandemic, ultraviolet-C (UV-C) decontamination of N95 respirators for emergency reuse has been implemented to mitigate shortages. However, decontamination efficacy across N95s is poorly understood, due to the dependence on received UV-C dose, which varies across the complex three-dimensional N95 shape. Robust quantification of UV-C dose across N95 facepieces presents challenges, as few UV-C measurement tools have sufficient 1) small, flexible form factor, and 2) angular response. To address this gap, we combine optical modeling and quantitative photochromic indicator (PCI) dosimetry with viral inactivation assays to generate high-resolution maps of "on-N95" UV-C dose and concomitant SARS-CoV-2 viral inactivation across N95 facepieces within a commercial decontamination chamber. Using modeling to rapidly identify on-N95 locations of interest, in-situ measurements report a 17.4 {+/-} 5.0-fold dose difference across N95 facepieces, yielding 2.9 {+/-} 0.2-log variation in SARS-CoV-2 inactivation. UV-C dose at several on-N95 locations was lower than the lowest-dose locations on the chamber floor, highlighting the importance of on-N95 dose validation. Overall, we couple optical simulation with in-situ PCI dosimetry to relate UV-C dose and viral inactivation at specific on-N95 locations to inform the design of safe and effective decontamination protocols.

Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2 (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

Characterization of cell-cell communication in COVID-19 patients (preprint)

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use it to examine five public single-cell RNA-seq datasets with a total of 85 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients.

Rapid detection of SARS-CoV-2 with Cas13 (preprint)

To combat disease outbreaks such as the COVID-19 pandemic, flexible diagnostics for rapid viral detection are greatly needed. We report a nucleic acid test that integrates distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We paired this workflow, termed Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER), with extraction-free sample lysis using shelf-stable reagents that are widely available at low cost. DISCoVER has been validated on saliva samples to incentivize frequent testing for more widespread community surveillance and robustly detected attomolar levels of SARS-CoV-2 within 30 minutes, while avoiding false positives in virus-negative saliva. Furthermore, DISCoVER is compatible with multiplexed CRISPR probes to enable simultaneous detection of a human gene control or alternative pathogens.

Practical considerations for Ultraviolet-C radiation mediated decontamination of N95 respirator against SARS-CoV-2 virus (preprint)

Decontaminating N95 respirators for reuse could mitigate shortages during the COVID-19 pandemic. We tested a portable UV-C light-emitting diode disinfection chamber and found that decontamination efficacy depends on mask model, material and location on the mask. This emphasizes the need for caution when interpreting efficacy data of UV-C decontamination methods.

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection (preprint)

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.