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.
