Kudo, T.; Meireles, A. M.; Moncada, R.; Chen, Y.; Wu, P.; Gould, J.; Hu, X.; Kornfeld, O.; Jesudason, R.; Foo, C.; Hockendorf, B.; Bravo, H. C.; Town, J. P.; Wei, R.; Rios, A.; Chandrasekar, V.; Heinlein, M.; Cai, S.; Lu, C. S.; Celen, C.; Kljavin, N.; Jiang, J.; Hleap, J. S.; Kayagaki, N.; Melo, F. d. S. e.; McGinnis, L.; Li, B.; Singh, A.; Garraway, L.; Rozenblatt-Rosen, O.; Regev, A.; Lubeck, E.

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated with in situ sequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NFKB signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in vivo screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.