Large-scale, spatially resolved panoramic CRISPR screening in native tissue environments using Perturb-DBiT.

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Spatially resolved in vivo CRISPR screening is limited in scale and scope.
• Current methods cannot comprehensively analyze perturbations in complex tissues.

Purpose of the Study:

• To develop a novel method for large-scale, spatially resolved CRISPR screening in situ.
• To investigate the impact of genetic perturbations on cancer metastasis and the tumor immune microenvironment.

Main Methods:

• Developed Perturb-DBiT for co-sequencing of spatial total RNA and single guide RNAs (sgRNAs) in situ.
• Applied large sgRNA panels (80,000+) to human cancer metastatic models and immune-competent mouse models.
• Integrated transcriptional pseudotime trajectories to analyze clonal dynamics.

Main Results:

• Linked perturbations in long noncoding RNAs, microRNAs, and tRNAs to tumor migration and growth.
• Observed significant impacts of perturbations on clonal dynamics and cooperation.
• Identified distinct synergistic effects on immune infiltration and suppression in the tumor microenvironment.

Conclusions:

• Perturb-DBiT offers a comprehensive, spatially resolved view of perturbation responses in complex tissues.
• The method facilitates understanding of small and large RNA regulation, tumor progression, and immune interactions.