Development of microfluidic enabled CRISPR-Cas9 functional genetic screening technologies for target discovery in cancer immunotherapy

Project Summary: Genome-scale genetic screens performed using CRISPR-Cas9 editing can interrogate determinants of cell viability and are powerful tools for the identification of genetic regulators. Using this technology, hundreds of millions of cells - each targeted with a specific genetic alteration - can be surveyed, typically based on a live/dead survival profiling. Phenotype-based genetic screens - where protein expression alterations are detected - represent a next-generation approach and can facilitate the identification of regulators of therapeutically-relevant proteoforms. Due to challenges related to implementation, phenotype-based screens are less commonly used compared to proliferation-based screens. Thus, rapid and robust selection approaches for targeted capture of live cells are required to realize the potential of phenotypic genome-scale screens for functional discovery, further annotation of the human genome, and discovery of novel targets for the development of therapeutics. Recently, through the use of a newly developed high-throughput approach for phenotypic CRISPR- Cas9 screening (Microfluidic Immunomagnetic Cell Sorting (MICS)) we processed an entire genome- wide screen containing more than 108 cells in under one hour to study factors that modulate the display of CD47 on the cell surface. This highly scalable cell sorting technology maintained high levels of cell viability throughout the screening process. CD47 is a widely expressed cell surface protein that acts as a “don’t eat me” signal through inhibitory interactions with SIRPa, a protein expressed on macrophages and other myeloid cells that negatively regulates phagocytosis. CD47 is highly expressed on various tumour types and blocking the CD47-SIRPa interaction has been explored as a novel cancer immunotherapy strategy that has shown promising results for some cancer types. We robustly identified modulators of CD47 function including QPCTL, an enzyme required for formation of the pyroglutamyl modification at the N-terminus of CD47 and interaction with SIRPa. The proposed study will expand the utility of the platform, develop new classes of microfluidic chips for cytometric analysis, and produce a comprehensive database of geno/phenotypic relationships. This new high-performance phenotypic assessment system will greatly accelerate target discovery for cancer therapeutics. In this project we will specifically interrogate VISTA (V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation) and cGAS/STING signaling pathways using a series of related cell lines to identify novel targets for immunotherapies. Importantly, these data will demonstrate the utility of the technology for rapid assessment of regulatory networks for drug discovery.