Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap.

Area of Science:

• Genomics
• Cell Biology
• Biotechnology

Background:

• Traditional sequencing methods for genetic screens require cell lysis, limiting the analysis of spatial phenotypes.
• Optical pooled genetic screens offer a way to study cellular responses, including morphology and tissue organization, to genetic perturbations.
• Investigating spatial phenotypes in diverse cell types and in vivo contexts remains challenging.

Purpose of the Study:

• To develop a multimodal optical pooled CRISPR screening method for in situ barcode readout.
• To enable the analysis of spatial phenotypes in challenging cell types and in vivo contexts.
• To apply the method to identify pathogenic mutations affecting cancer therapy response.

Main Methods:

• CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection.
• Barcode detection is enhanced through combinatorial hybridization of DNA oligos.
• The method allows for in situ readout in various cell types, including primary cells, stem cells, neurons, and in vivo tissues.

Main Results:

• CRISPRmap successfully enabled in situ barcode readout in previously inaccessible cell types and contexts.
• A screen in breast cancer cells revealed the effects of DNA damage repair gene variants on cellular responses to cancer therapies.
• Optical phenotyping using CRISPRmap identified likely pathogenic patient-derived mutations previously classified as variants of unknown clinical significance.

Conclusions:

• CRISPRmap is a powerful multimodal screening platform for in situ genetic perturbation analysis.
• The method expands the applicability of optical pooled screening to diverse biological systems.
• CRISPRmap has clinical relevance in identifying pathogenic mutations for improved cancer therapy selection.