Spatially Resolved Panoramic in vivo CRISPR Screen via Perturb-DBiT

Area of Science:

• Genomics
• Molecular Biology
• Systems Biology

Background:

• Current in vivo CRISPR screening methods have limitations in perturbation panel size and RNA detection.
• Existing spatial transcriptomics approaches struggle to integrate genetic perturbations with whole-transcriptome analysis.
• Understanding genetic perturbation effects in native tissue environments requires advanced spatial resolution.

Purpose of the Study:

• To develop a versatile approach for simultaneous spatial whole-transcriptome and single-guide RNA (sgRNA) co-sequencing.
• To enable unbiased discovery of how genetic perturbations influence RNA regulation, cellular dynamics, and tissue architecture in situ.
• To provide a comprehensive, spatially resolved view of genetic knockout impacts on diverse molecular and cellular responses.

Main Methods:

• Development of Perturb-DBiT, a novel method for simultaneous co-sequencing of spatial total RNA and sgRNAs on the same tissue section.
• Application of Perturb-DBiT to human cancer metastatic colonization and immune-competent syngeneic mouse models.
• Integration of transcriptional pseudotime trajectories with spatial transcriptomic data.

Main Results:

• Mapped large panels of sgRNAs and corresponding total RNA transcriptomes in human cancer models, revealing insights into lncRNA co-variation, microRNA-mRNA interactions, and tRNA alterations.
• Uncovered the impact of perturbations on clonal dynamics and cooperation using transcriptional pseudotime analysis.
• Investigated genetic perturbations in the tumor immune microenvironment, demonstrating distinct and synergistic effects on immune infiltration and suppression.

Conclusions:

• Perturb-DBiT provides a powerful, spatially resolved platform for comprehensive analysis of genetic perturbations in complex tissues.
• The method enables unbiased discovery of molecular and cellular responses, including RNA regulation, tumor progression, and immune interactions.
• Perturb-DBiT offers a panoramic perspective on perturbation effects, advancing our understanding of gene function in vivo.