Spatially Resolved in vivo CRISPR Screen Sequencing via Perturb-DBiT

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Perturb-seq profiles gene expression changes from genetic perturbations in single cells.
• However, it cannot assess the impact of these perturbations on the tissue microenvironment.

Purpose of the Study:

• To develop Perturb-DBiT, a novel platform for simultaneous spatial transcriptome and guide RNA (gRNA) co-sequencing.
• To enable in vivo CRISPR screens with genome-scale gRNA libraries for comprehensive analysis of genetic modifications in tissue contexts.

Main Methods:

• Perturb-DBiT integrates spatial transcriptomics with CRISPR screening by co-sequencing gRNAs and transcriptomes from the same tissue section.
• The platform accommodates various delivery vectors, gRNA library sizes, tissue preparations, and gRNA capture methods.
• Genome-scale CRISPR screens were performed across three cancer models, including a human metastatic colonization model and an immune-competent syngeneic model.

Main Results:

• Perturb-DBiT mapped gRNAs and transcriptomes in individual colonies, revealing clonal dynamics and cooperation in cancer metastasis.
• The study uncovered how genetic perturbations differentially and synergistically affect immune cell infiltration and suppression within the tumor microenvironment.
• The method demonstrated the ability to simultaneously assess knockout effects on tumor initiation, development, metastasis, histopathology, and immune landscape.

Conclusions:

• Perturb-DBiT significantly expands the scope of genetic inquiry by integrating spatial and functional genomics.
• This platform provides a comprehensive understanding of genetic modifications' effects on cellular behavior and tissue architecture.
• Perturb-DBiT lays the foundation for developing targeted therapeutic strategies by elucidating complex gene-environment interactions in cancer.