High-content optical pooled genome-wide screens of SARS-CoV-2 infection

PROJECT SUMMARY Identifying gene function and impact on disease biology are overarching aims of life science research in the post-genomic era. Functional genomics also underpins our ability to understand the meaning of genetic variation inhuman populations. However, crucial gaps remain in the functional genomics tool set that will slow our progress in applying genomics to unravel disease biology. Currently, efficiently pooled methods for genome-wide screening require either selection of cells based on growth advantage or physical purification (e.g. by FACS orfor single-cell analysis). Many disease processes are characterized by complex cellular phenotypes including defects in cell or organelle morphology, subcellular localization of molecular components, or cell motility. Other key phenotypes of interest may involve transient states (eg mitosis), cell-cell interactions, or require dynamicassays in live cells (eg, optical recording of electrophysiological activity of cardiac or neural cells). Image-based,high-content screens using overexpression and RNA interference have uncovered novel genes involved incomplex phenotypes, including mitosis, synaptogenesis, and embryogenesis. However, such microplate-based screens of clonal cell populations are not regularly conducted at the genomic scale due to the expense, labor,and automation expertise required. In this program, we developed a new genomic perturbation and screening concept that combines major advantages of pooled perturbation with imaging assays for single-cell arrayedreadout of complex phenotypes. Specifically, we screen pooled genomic perturbations (with CRISPR-Cas9single-guide RNAs) using microscopy to read out phenotypes AND to identify perturbed genes at the single-cell level via in situ sequencing with a sequencing by synthesis approach. This approach is highly scalable because reagent and instrumentation costs are modest (now a few tens of thousands of dollars for a genome-widescreen). Here we request an administrative supplement to apply the technology developed in our existing award to screens for SARS-CoV-2 infection of cell lines with Rob Davey's group at the Boston University NortheastEmerging Infectious Disease Laboratory that is equipped and actively working with high-containment viral pathogens including SARS-CoV-2. This work on antiviral host cell programs is within the scientific scope of the original grant. We will tightly coordinate the rapid execution of optical pooled screens in multiple biological models with Dr. Davey's ongoing conventional CRISPR genomic screening activity. The data-rich genome-wide opticalscreening data will identify new aspects of the SARS-CoV-2-host interface across the viral life cycle and advance our understanding of candidate therapeutics as well as support the generation of new therapeutic hypotheses to address the COVID-19 pandemic.