Abstract

Cell biological states are defined by the structural and molecular characteristics of cells determined through a combination of genetic and epigenetic factors responsive to the cellular environment. Microscopy remains the most direct and scalable approach to explore the intricate spatio-temporal complexity of cells in both simple research models and multicellular organisms. Innovation in imaging tools continues to expand the achievable depth and specificity of single-cell molecular characterization using microscopy, and can now be leveraged for large-scale pooled genetic screens with deep single-cell profiling capacity. This combination is revealing how biological systems work in detail and at scale by cataloging many cellular phenotypes with clear causal relationships in large experimental batches. I will introduce this class of technology and explain how my group recently combined pooled CRISPR/Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules to reveal measurable phenotypes for most essential gene knockouts. We clustered knockout phenotypes based on hundreds of quantitative parameters to group genes across a wide spectrum of specific cellular processes, reveal co-functional genes, and provide predictions for gene functions. Our group is now working to expand the ranges of perturbation types, phenotyping assays, and biological systems compatible with this approach.