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Revolutionizing Gene Function Analysis with PERISCOPE

Understanding the role of specific genes often requires the visualization of cellular changes following genetic modifications. While microscopy offers valuable insights, the challenge lies in its scalability when dealing with extensive gene studies involving thousands of targets simultaneously.

Researchers from the Broad Institute of MIT and Harvard, in collaboration with Calico Life Sciences, have introduced a transformative approach that enhances the application of microscopy in genome-scale CRISPR screenings. This method, known as PERISCOPE (perturbation effect readout in situ via single-cell optical phenotyping), merges two innovative techniques: Cell Painting and Optical Pooled Screening.

Cell Painting allows for the simultaneous imaging of multiple subcellular structures, utilizing distinct colors to represent various organelles, such as blue for nuclei and magenta for the endoplasmic reticulum. In conjunction with Optical Pooled Screening, which leverages CRISPR-based barcoding techniques to systematically disable genes, PERISCOPE enables comprehensive examinations of over 20,000 genes and their influence on various cellular characteristics.

This novel technique is reportedly more than ten times less expensive than other high-dimensional methodologies, like high-throughput single-cell RNA sequencing. Moreover, it can be applied to a broad spectrum of cell types, underscoring its versatility in genetic research.

In their recent publication in Nature Methods, the team showcased the application of PERISCOPE through three extensive whole-genome CRISPR screens, creating an open-access atlas that connects cell morphology to gene function.

This pioneering study was spearheaded by a team of prominent scientists, including JT Neal, Anne Carpenter, and Paul Blainey from the Broad Institute, and Calvin Jan from Calico. The collaborative effort also saw contributions from scientists like Meraj Ramezani, Erin Weisbart, Julia Bauman, and Avtar Singh, all of whom played key roles in developing the research.

“This atlas represents a groundbreaking genome-scale resource for linking cellular structure to gene function,” stated Neal. “A significant advantage of this project is that all generated data is open access, complemented by robust analysis pipelines that serve the scientific community for analyzing optical pooled screening data at scale.”

Advancing Cellular Research through Morphological Insights

Originally conceptualized by Anne Carpenter in 2013, the Cell Painting assay has evolved significantly, enabling simultaneous imaging of multiple cellular organelles and providing extensive data on cellular morphology. Advanced machine-learning algorithms analyze the images, detecting subtle variations in cellular texture and other morphological features, crucial for assessing gene disruption effects.

When Neal joined the Broad Institute in 2017, he was motivated by the potential of employing Cell Painting for cancer cell research to identify key genes and variants linked to targeted cancer therapies. Collaborative discussions with Carpenter and Blainey catalyzed the integration of Cell Painting with Optical Pooled Screening, leading to the conception of PERISCOPE in 2019.

In the PERISCOPE framework, a library of guide RNAs targeting approximately 20,000 genes is introduced into cells, followed by the activation of the CRISPR Cas9 enzyme to inactivate these genes. This process generates complementary DNA from the guide RNAs, effectively barcode each gene knockout, thus allowing researchers to analyze multiple gene modifications within a single experimental setup.

The imaging process employs standard widefield microscopy to capture detailed images of the various cell structures stained with the five colors from the Cell Painting process, along with the barcode identifications for the targeted genes. Subsequent automated analyses facilitate the extraction of phenotypic data, establishing a correlation between cellular characteristics and gene activity.

Utilizing PERISCOPE, the research team developed atlases detailing the effects of gene knockouts in human lung and cervical cancer cells. These comprehensive resources not only confirmed existing biological knowledge but also unveiled novel insights into poorly characterized genes. Notably, the function of TMEM251—a gene implicated in a rare lysosomal storage disorder—was elucidated, highlighting its importance in enzyme trafficking to lysosomes.

The research team is now focused on enhancing PERISCOPE’s capabilities by increasing the number of colors used in simultaneous imaging, thereby broadening the range of cellular traits that can be assessed. Additionally, they are collaborating with other researchers to expand the application of PERISCOPE, targeting projects in cardiometabolic diseases and exploring potential utilities in neurodegenerative conditions like Parkinson’s disease.

Neal noted, “Historically, studying genetic interactions has presented challenges due to the rapidly growing complexity of gene interaction networks. However, with PERISCOPE and scalable optical pooled screening techniques, the prospect of conducting extensive genetic interaction studies is not only feasible but close at hand.”

Further Reading: Ramezani M et al., “A genome-wide atlas of cell morphology,” Nature Methods (2025). DOI: 10.1038/s41592-024-02537-7.

For more information, visit the Broad Institute of MIT and Harvard’s official site.

Source
phys.org