Uncovering spatially resolved functional genomics with CRISPR screen sequencing.

Spatial omics has advanced our understanding of tissue-level biology, yet tools to systematically link gene functional perturbations to spatial phenotypes and signaling pathways remain limited. To address this, we developed spatial CRISPR screen sequencing (SPAC-seq), a high-throughput spatial CRISPR screen platform, and TARDIS (target prioritization toolkit for perturbation data in spatial omics), a statistical spatial perturbation analysis toolkit. Using SPAC-seq and TARDIS, we linked gene perturbations to spatial phenotypes and pathways, uncovering how Icam1 loss in tumor cells promotes metastasis via immune suppression and macrophage polarization. In CD8+ T cells, we revealed Cd44's role in regulating spatial phenotypes by interacting with Spp1 on macrophages. We also demonstrated the model of the transcription factor-chemokine receptor axis coupling cell states with chemotaxis. SPAC-seq and TARDIS provide an effective framework to study spatially resolved functional genomics and pathways across diverse biological and disease contexts.