Optical pooled screening (OPS) is a powerful tool for linking morphological cellular phenotypes with in situ readouts of genetic perturbations and has been used to identify the regulators of diverse biological processes. Adoption of OPS has been restricted to a small number of laboratories due to the sophisticated expertise required for data generation and computational analysis.
Here, we establish a fully automated end-to-end workflow for OPS on the AVITI24™ platform. See how we demonstrate highly accurate in situ sequencing of genetic perturbations combined with a highly multiplexed AVITI24 phenotyping panel (up to 50 proteins and 350 mRNAs) and apply the combined protocol to perform a 500-gene CRISPR screen for regulators of complex cellular processes. This work lays the foundation for automated OPS data generation, supporting rich profiling of perturbation effects at unprecedented scale.