Simultaneous CRISPR screening and spatial transcriptomics reveals intracellular, intercellular, and functional transcriptional circuits.

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.