In situ functional genomics to understand transcriptional regulation

PROJECT SUMMARY The COVID-19 pandemic has taken the lives of nearly 500,000 people worldwide in the span of a few months.Recently, a novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) hasemerged and rapidly surpassed others in prevalence, including the original SARS-CoV-2 isolate from Wuhan,China. This Spike variant is a defining feature of the most prevalent clade (A2a) of SARS-CoV-2 genomesworldwide and, recently, we and others have demonstrated this variant leads to virions with an ~8-fold increasein human cell transduction. This is the first experimental evidence of a SARS-CoV-2 population variant acting ina gain-of-function manner. Although there are hundreds of Spike variants now in circulation, we lack tools for high-throughputcharacterization of these variants and their virulence. Here, we propose to develop a massively-parallel, high-throughput approach to test all Spike variants using a pooled forward genetic screen, examine the impact ofthese mutations on proteolytic cleavage of Spike and on ACE2 receptor binding kinetics, and validate changesin viral transduction with live SARS-CoV-2 via an innovative trans-complementation assay. Our proposed studies aim to understand the interactions between Spike protein variants and host (human)cell infection and their underlying biochemical mechanisms. This research will enable us to predict whetherparticular Spike variants can drive more serious COVID-19 outbreaks.