Spatially Resolved Panoramic in vivo CRISPR Screen via Perturb-DBiT.

Spatially resolved in vivo CRISPR screening integrates gene editing with spatial transcriptomics to examine how genetic perturbations alter gene expression within native tissue environments. However, current methods are limited to small perturbation panels and the detection of a narrow subset of protein-coding RNAs. We present Perturb-DBiT, a distinct and versatile approach for the simultaneous co-sequencing of spatial total RNA whole-transcriptome and single-guide RNAs (sgRNAs), base-by-base, on the same tissue section. This method enables unbiased discovery of how genetic perturbations influence RNA regulation, cellular dynamics, and tissue architecture in situ. Applying Perturb-DBiT to a human cancer metastatic colonization model, we mapped large panels of sgRNAs across tumor colonies in consecutive tissue sections alongside their corresponding total RNA transcriptomes. This revealed novel insights into how perturbations affect long non-coding RNA (lncRNA) co-variation, microRNA-mRNA interactions, and global and distinct tRNA alterations in amino acid metabolism linked to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further uncovered the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, Perturb-DBiT enabled investigation of genetic perturbations within the tumor immune microenvironment, revealing distinct and synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of how genetic knockouts influence diverse molecular and cellular responses including small and large RNA regulation, tumor proliferation, migration, metastasis, and immune interactions, offering a panoramic perspective on perturbation responses in complex tissues.