RAPID: Comparative CRISPR Genetic Screens to Understand Zoonotic Transmission of SARS-CoV-2

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To date the COVID-19 pandemic has infected over 3,000,000 people and resulted in approximately 200,000 deaths worldwide. Viruses rely upon their target cells to provide the proper machinery for their life cycle. Seeing as the coronavirus (SARS-CoV-2) responsible for COVID-19 can infect both bats and humans, this project aims to identify common and unique molecular interactions required for infection in both organisms. In response to the NSF’s “Dear Colleague Letter” this proposal uses cutting-edge genetic tools such as CRISPR/Cas9 to gain knowledge about cellular factors needed for virus replication. Throughout the course of this research, genetic tools to identify these interactions in bat cells will be created, providing a resource for any researcher who wishes to study these interactions further. This information will also inform researchers on the specific interactions necessary for coronavirus replication that are common between bat and human cells, providing a framework to fully understand this SARS-CoV-2, in addition to future, undiscovered pathogens. Molecular interactions that are unique to bat and human cell lines provide insight into what interactions are overcome for survival in multiple species and also may provide insight into how an infection can be benign in one species (bats) and cause disease in another (humans). This project will discover new knowledge essential to coronavirus biology, providing the basis for future in depth studies. In addition to increasing knowledge about SARS-CoV-2 biology, this proposal also supports the training of a post-doctoral fellow, broadening participation in STEM. The zoonotic transmission of SARS-CoV-2 from the horseshoe bat to humans represents a large question in coronavirus biology. What are the host-virus interactions and cellular pathways in common between bats and humans that allow for efficient replication and virus survival in both species? To answer this question, CRISPR/Cas9 will be used to effectively knockout every gene in both bat and human cell lines to identify genes are required for coronavirus replication in each cell line. While CRISPR/Cas9 is a powerful genetic tool, currently these tools are limited to the study of human and murine models, representing a significant challenge for the study of viral replication in bat cell lines. A novel computational pipeline named “MerryCRISPR” will be used to design bat CRISPR reagents and subsequently build the tools necessary for this work. The combination of whole host genome-viral interactions studies studied in parallel in both bat and human cell lines will provide the necessary information to build functional and comprehensive host-coronavirus interaction maps in addition to identifying species-specific interactions. Collectively, these studies will provide the basis for future interrogation of specific and unique host-coronavirus interactions, informing researchers on the essential biology of coronaviruses. This RAPID award is made by the Physiological and Structural Systems Cluster in the BIO Division of Integrative Organismal Systems, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.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.