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New Approach to Gene Function Exploration: CRISPRmap
Researchers from the Gaublomme Lab have introduced an innovative optical pooled screening technique named CRISPRmap. This method integrates the optical characteristics of individual cells with targeted genetic modifications, enabling scientists to access critical phenotypic information that traditional sequencing methods often overlook. These optical traits, which consist of cell morphology, protein localization, cell-cell interactions, and the organization of tissue, are essential for understanding cellular behavior in various biological contexts.
CRISPRmap facilitates a detailed examination of gene functions within tissue, granting researchers insight into both intrinsic and extrinsic cellular responses that are typically unavailable in in vitro experiments. This breakthrough could significantly enhance our comprehension of how certain genes influence processes like immune cell recruitment to tumors, metastasis, invasion, and angiogenesis.
The findings were recently published in Nature Biotechnology. The CRISPRmap methodology enables high-throughput genetic investigations by allowing the simultaneous analysis of numerous cells subjected to various genetic perturbations. Each participating cell expresses a unique RNA barcode identifying the specific CRISPR alteration that has taken place within it.
Importantly, CRISPRmap excels in contexts previously challenging for other methodologies, such as stem cells and in-vivo cells within complex tissue environments. Once a perturbed gene is identified in a specific cell, researchers can assess how that cell and its surrounding environment react.
“Our lab has tailored CRISPRmap to function with optical readout assays, which supports concurrent multiplexed profiling of proteins and RNA species,” noted Professor Jellert Gaublomme, the primary author of the study. “Additionally, CRISPRmap is versatile in terms of genetic perturbations, paving the way for experiments involving targeted mutations, gene interference and activation, epigenetic modifications, and CRISPR RNA editing.”
In collaboration with the Ciccia lab at Columbia University Irving Medical Center, the team employed CRISPRmap to explore the functional impacts of 292 mutations across 27 genes integral to the DNA damage response mechanism. This involved visualizing how DNA repair proteins are recruited to sites of DNA damage, particularly following exposure to ionizing radiation or common chemotherapeutic agents utilized in breast cancer treatment.
The researchers meticulously profiled the expression and localization patterns of various proteins and mRNA species across around one million cells, thus enabling a refined analysis of how gene variants influence the DNA damage response. “This methodology helped us pinpoint missense variants of uncertain clinical relevance that exhibit responses akin to known pathogenic variants. Consequently, our approach can serve as a valuable framework for annotating human variants in a context-sensitive manner, assisting in the prioritization of therapeutic strategies,” explained Jiacheng Gu, the lead author of the study.
Moving beyond the confines of cancer cell lines in laboratory settings, the team successfully demonstrated CRISPRmap barcode detection in cancer cells within the tumor microenvironment—a primary focus of the NIH Director’s New Innovator Award awarded to the lab.
In collaboration with the Chan lab, the research group utilized CRISPRmap to analyze tumor sections, combining optical barcode data with multiplexed antibody staining to visualize processes such as angiogenesis and extracellular matrix formation around tumor domains. This enhanced understanding of transcription factor nuclear translocation among transplanted cells further showcases the capabilities of CRISPRmap.
CRISPRmap’s adaptability across different CRISPR technologies and cellular contexts positions it as a versatile tool applicable to various biological and medical studies. “We aimed to optimize our technique for widespread use, as it does not depend on third-party sequencing reagents for barcode reading. Moreover, the readout dyes can be tailored to fit the microscopy equipment researchers have at their disposal, making the approach both flexible and economically viable,” noted Gu.
“We foresee that researchers will leverage this to conduct perturbation studies relevant to their specific cell types, allowing exploration of biological pathways at a scale comparable to the number of genes implicated in those pathways,” added Gaublomme.
Looking ahead, the Gaublomme lab intends to investigate how gene modifications affect tissue architecture and cell interactions within complex microenvironments. There are plans to extend research to patient-derived organoids, facilitating the exploration of gene functions within specific disease contexts.
“We envision CRISPRmap as a means to uncover optical phenotypes across a spectrum of biological scales, from molecular domains such as DNA damage sites within individual nuclei to wider cellular reorganizations impacting entire organs,” concluded Gaublomme. “The applications of this approach could span from fundamental biology to understanding disease mechanisms and refining therapeutic strategies in fields ranging from cancer to neurodegenerative disorders.”
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
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Citation: Scientists develop a new method to study gene function in cells and tissue (2024, October 14) retrieved 14 October 2024 from https://phys.org/news/2024-10-scientists-method-gene-function-cells.html
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