Scientists develop a new method to study gene function in cells and tissue

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The Gaublomme lab has developed a new optical pooled screening approach called CRISPRmap, which enables the coupling of optical properties of single cells to targeted genetic perturbations. Optical phenotypes are typically inaccessible for sequencing-based approaches based on cell lysis but include crucial information such as cell morphology, protein subcellular localization, cell-cell interactions, extracellular matrix factors, and tissue organization.

CRISPRmap allows for spatially resolved interrogation of gene function in tissues, enabling researchers to map both cell-intrinsic and cell-extrinsic effects of perturbations, which are not accessible through in vitro studies. This approach opens new avenues for understanding genes involved in immune cell recruitment to tumors, metastasis, invasion, angiogenesis, etc.

The Gaublomme group shared their findings in a study recently published in Nature Biotechnology.

Performing these studies in a pooled fashion enables high-throughput genetic studies by measuring the responses of many cells to different genetic perturbations in parallel. In pooled analyses, each cell expresses RNA transcripts that encode a barcode that identifies which CRISPR perturbation occurred in that cell.

Notably, CRISPRmap enables optical barcode readout in challenging cell types and contexts previously elusive to other methods, including stem cells, derived neurons, and in-vivo cells within tissue contexts. After identifying which gene is perturbed in a given cell, scientists can learn about the response of cells and their environment to this perturbation.

"Our lab has optimized CRISPRmap to be compatible with optical readout assays, allowing concurrent multiplexed profiling of proteins and mRNA species. Moreover, CRISPRmap is agnostic to the type of genetic perturbation, opening the way to explore targeted mutations, gene interference and activation, epigenetic modification, and CRISPR RNA editing," explained Professor Jellert Gaublomme, the study's corresponding author.

In collaboration with the Ciccia lab at Columbia University Irving Medical Center, the authors applied CRISPRmap to investigate the functional consequences of 292 mutations in 27 genes critical for the DNA damage response, visualizing the recruitment of DNA damage repair proteins to DNA damage sites.

They assessed these responses after ionizing radiation or DNA-damaging agents commonly used as chemotherapeutic drugs in breast cancer. Profiling the expression and subcellular localization of dozens of proteins and mRNA species in approximately one million cells allowed for a nuanced interrogation of variant influences on the DNA damage response.

"This method allowed us to identify missense variants of uncertain clinical significance whose response resembles known pathogenic variants. As such, our approach can provide a framework for annotating human variants in a treatment-specific manner and help prioritize therapeutic strategies," described Jiacheng Gu, the study's first author.

Beyond studies of cancer cell lines grown in a dish, the researchers demonstrated their CRISPRmap barcode detection in cancer cells in the tumor microenvironment, a key goal of the NIH Director's New Innovator Award that the lab received.

Collaborating with the Chan lab, they profiled tumor sections with CRISPRmap barcode detection and combined the readout with multiplexed antibody staining to visualize angiogenesis, extracellular matrix formation around tumor domains, and transcription factor nuclear translocation in the transplanted cells.

As CRISPRmap is applicable across various CRISPR modalities and cell types, the technique can be utilized for a wide range of research in biology and medicine. "We optimized our approach for broad accessibility since it does not rely on third-party sequencing reagents for barcode detection, readout dyes can be customized to match microscopes available to researchers, and the approach is cost-effective," explained Gu.

"We envision this will further enable individual labs to perform perturbation studies in their cell type of interest, probing biological pathways at a scale similar to the number of genes known to play a role in the pathway. " added Gaublomme.

The Gaublomme lab plans to explore the impact of gene perturbations on tissue architecture and the interplay between cells in complex microenvironments. Further studies could focus on patient-derived organoids to study gene function in tissue and disease-specific contexts.

"We envision CRISPRmap to elucidate optical phenotypes across a wide range of biological length scales, from molecular scales such as DNA damage break foci in a single nucleus to cellular reorganization across whole organs.

"The approach could be applied to studies that range from basic biology to disease mechanisms and optimization of therapeutic approaches in development, neurodegenerative diseases, and cancer," concluded Gaublomme.

More information: Jiacheng Gu et al, Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap, Nature Biotechnology (2024). DOI: 10.1038/s41587-024-02386-x

Journal information: Nature Biotechnology

Provided by Columbia University