Highlights

• Perturb-FISH integrates spatial transcriptomics with CRISPR perturbation detection to map genetic effects in single cells.

• Captures cell-specific transcriptional responses to genetic perturbations, aligning with Perturb-seq findings in immune-challenged monocytes.

• Reveals spatial genetic networks influenced by cell proximity and tissue density in innate immunity.

• Identifies altered tumor-immune interactions in 3D xenograft models following genetic knockout.

• Links autism risk gene perturbations in hiPSC astrocytes to calcium signaling defects and dysregulated pathways.

Summary
Perturb-FISH is a novel approach that merges spatial transcriptomics with optical detection of CRISPR guide RNAs, enabling simultaneous analysis of genetic perturbations and their molecular consequences at single-cell resolution. Validating its precision, the method replicates intracellular transcriptional effects observed in Perturb-seq during a monocyte lipopolysaccharide response screen, while also uncovering intercellular communication and tissue density-dependent immune regulation. Applied to 3D xenograft tumors, Perturb-FISH exposes how genetic knockouts reshape tumor-immune microenvironment interactions. In a parallel screen of autism spectrum disorder risk genes using human iPSC-derived astrocytes, the method connects CRISPR perturbations to shared calcium signaling abnormalities and pinpoints associated genetic networks and dysregulated pathways. By bridging spatial transcriptomics, CRISPR screens, and functional phenotyping, Perturb-FISH emerges as a versatile platform for dissecting the genetic, spatial, and functional interplay driving cellular behavior in complex systems.
Fulltext
https://www.cell.com/cell/fulltext/S0092-8674(25)00197-7