Beyond Static Screens: A High-Throughput Pooled Imaging CRISPR Platform for Dynamic Phenotype Discovery

Understanding the dynamic regulation of signaling pathways requires methods that capture cellular responses in real time. While high-content imaging-based genetic screens have transformed functional genomics, they have remained largely limited to static or binary phenotypes. Here, we present DynaScreen, an imaging-based, pooled CRISPR screening platform that enables high-throughput investigation of dynamic cellular phenotypes at single-cell resolution. By integrating Forster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) biosensors with photoactivation-based single-cell tagging and pooled CRISPR screening technology, we establish a scalable system to identify genes that regulate the timing, amplitude, and duration of signaling responses. As proof of principle, we applied this approach to the cAMP signaling pathway, a key regulator of cellular physiology. Using a custom guide RNA (gRNA) library, we tracked real-time cAMP dynamics in response to agonist stimulation and identified genes that modulate its basal levels and response kinetics. Cells with aberrant signaling were selectively photoactivated, isolated by fluorescence-activated cell sorting (FACS), and subjected to next-generation sequencing to pinpoint causal genetic perturbations. This strategy successfully uncovered known and novel regulators of cAMP dynamics. In conclusion, the integration of FLIM microscopy, CRISPR technology and open-source software to handle image analysis, automated hit identification and data representation, enables real-time exploration of dynamic phenotypes in a wide range of biological settings.