Automation and High-Throughput Technologies
Advanced imaging technologies to bridge the gap between high-content and high-throughput
Pooled genetic screens have been critical for the systematic identification of genes underlying cellular processes, but have thus far been limited to phenotypes defined by cellular enrichment or comparatively low-throughput single-cell molecular profiling. We have developed a method to make pooled libraries compatible with the rich set of spatially and temporally resolved phenotypes accessible to high-content microscopy by using targeted in situ sequencing to demultiplex genetic perturbations. We applied this technology to screen 952 genes for involvement in NF-κB signaling by imaging p65 nuclear translocation and relaxation, recovering most canonical pathway members and identifying novel candidate regulators of IL-1β/TNFα-stimulated immune responses. We are currently piloting applications with a range of optical assays and cell models and expect that pooled optical screens will have broad utility in identifying genetic components, analyzing genetic circuits, and interrogating disease variants.