CRISPR Screening in Expanded Phenotypic Space

Abstract

The use of CRISPR-Cas systems in genetic screening has permitted rapid functional annotation of the genome. However, conventional pooled CRISPR screening is constrained in several ways, including: (1) limited diversity of biomolecules and biological functions which can be investigated; (2) limited ability to resolve phenotypes over time; and (3) the high-throughput nature of pooled CRISPR screening demands that a small number of parameters are considered, making CRISPR screening ‘low content’. Several examples of CRISPR screens designed to circumvent these limitations have been reported. In this thesis we define these approaches, which expand the phenotypic space in which pooled CRISPR screens can be performed, as expanded phenotypic screens (EPS). We sort these EPS into categories based on commonalities in experimental design. We then apply EPS to investigate biological functions which cannot be probed with conventional pooled CRISPR screening approaches. We first report the use of fluorophore-conjugated SARS-CoV-2 Spike protein in a FACS-based CRISPR activation screen to identify novel SARS-CoV-2 Spike-binding proteins. This screen identified LRRC15, which we validated as being expressed in the human lung in the context of COVID-19, acting to inhibit SARS-CoV-2 infection in trans and increasing antiviral and antifibrotic tone. We then used the fluorophore-tagged cholesterol-binding domain 4 of anthrolysin O (ALOD4-Neon) in a FACS-based CRISPR knockout screen to identify novel regulators of plasma membrane cholesterol abundance. Finally, we developed a novel workflow for FACS-based detection of increased cytosolic Ca2+ and applied this system in a proof-of-concept CRISPR activation screen to identify novel regulators of capsaicin-induced Ca2+ flux. These applications demonstrate the power of EPS in yielding candidate hit genes with previously uncharacterised roles that will be subject to further validation and considered for development of therapeutics.