2025 Theses Doctoral

Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap

Gu, Jiacheng

Pooled CRISPR screening has become a widely adopted high-throughput approach for studying gene functions by measuring cellular responses to diverse genetic perturbations in parallel. Integrating single-cell RNA sequencing with pooled CRISPR screens enabled the measurement of transcriptomic responses to genetic perturbations at single-cell level. However, such approaches inherently require cell isolation and lysis, making them unable to capture spatial organization of intercellular or intracellular phenotypes.

To address this limitation, imaging-based opitcal pooled CRISPR screens have emerged to enable the capture of complex cellular morphology and spatially resolved molecular phenotypes. I, under the supervision of my mentor Dr. Jellert Gaublomme and together with my colleagues in the Gaublomme Lab and our collaborators, implemented the design of CRISPRmap, a sequencing-free optical pooled CRISPR screening approach. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context.

Notably, I conducted optical base-editing screens in a breast cancer cell line to investigate the effects of DNA damage response gene variants on cellular responses to commonly used cancer therapies, and showed that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.