RAPID:CRISPR Whole genome screen to identify cellular machinery and pathways of SARS-CoV-2 entry into cells

This project will discover genes that help and/or inhibit the entry of the COVID-19 virus into cells using a new unbiased screening approach. This work will benefit society by creating a more comprehensive view of how COVID-19 infects cells. In addition, this work has the potential to identify host cell antiviral defense genes that can inhibit viral entry, as well as define genes that contribute to antibody protection in immune cells. Here, new CRISPR technology will be applied to screen the entire human genome to find these genes by correlating gene inactivation with increased or decreased viral entry into lung and immune cells. This information will provide the knowledge needed to develop new anti-viral strategies and to understand how environmental, genetic and cellular factors may influence the susceptibility of an individual to infection as well as the severity of the infection. The Broader Impacts of the project include training the next generation of virologists and cell biologists. Graduate students will participate in all phases of the project, including learning state-of-the-art CRISPR technologies. With the help of high school teachers, the work of this project will also be used to produce artistic descriptions that will allow broad dissemination of our findings to students, the scientific community, and the broader public.

This work aims to elucidate host genes that promote and inhibit the entry of the SARS-CoV-2 virus, the causative agent of COVID-19, into epithelial cells and macrophages using new whole-genome CRISPR screening approaches. At present, the ACE2 viral receptor and the host cell protease, TMRPSS2, have been the focus of research on the machinery needed for viral entry. However, other receptors, including CD147 and other proteases can facilitate viral entry indicating that a broader perspective is needed. Conversely, little is known about the role of innate antiviral genes that may restrict viral entry. Rapid adaptation of current CRISPR whole genome screening strategies will enable identification and ranking of all genes contributing to and inhibiting SARS-CoV-2 entry into epithelial cells expressing the ACE2 receptor and macrophages via antibody/Fc receptor or CD147-mediated attachment. Screens conducted in epithelial cells are expected to provide a map of the genes that contribute to and inhibit infection as well as viral spread within the lung. While it is not yet clear the extent to which SARS-CoV-2 can replicate in macrophages, multiple lines of evidence indicate that the virus can infect macrophages in a process aided by the presence of antibodies that may modulate their inflammatory state contributing to COVID-19. The screening strategy of the Project using pseudotyped lentiviral vectors will enable detection of viral entry, absent viral replication, and will provide a description of the genes aiding and inhibiting antibody-dependent viral entry into macrophages. Comprehensive genetic analysis across both cell types will provide an unprecedented view of viral entry and will provide scientific knowledge urgently needed to combat COVID-19.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.