PCSK9 is a secreted enzyme that plays an important role in controlling the amount of LDL receptor activity in the liver, which in turn influences the levels of LDL in the bloodstream and their contribution to atherosclerotic diseases including heart attack and stroke. 

In 2018, we applied genome-scale CRISPR screening to identify SURF4 as an ER cargo receptor that mediates PCSK9 secretion of PCSK9. Subsequent work has been focused on confirming the physiologic relevance of this discovery by generating and characterizing mice with germline and liver-specific Surf4 deletion, and on defining the full repertoire of SURF4 cargoes. 

Because of the important influence of hepatic LDL receptor activity on atherosclerosis, we next extended our application of CRISPR screening to directly query this process. 

In 2021, we reported our genome-scale CRISPR screen for functional modifiers of cellular LDL endocytosis. In addition to canonical LDLR regulators, we identified dozens of new genes whose disruption influenced the amount of fluorescent LDL internalized by liver-derived cells. We synthesized a custom secondary CRISPR library for high-resolution validation of our primary screen and for orthogonal testing to clarify the mechanism, specificity, and generalizability of each gene. Subsequent work has been focused on a deeper characterization of select novel LDLR regulators including the GTPase RAB10 and the transcription factor PROX1.  

At the onset of the COVID-19 pandemic, we applied our expertise in high-throughput CRISPR genome editing to screen for genes relevant to SARS-CoV-2 biology. We first developed a flow cytometry-based assay for quantifying the abundance of ACE2 (the receptor for SARS-CoV-2 entry) on the surface of host cells, revealing an unexpected heterogeneity in ACE2 expression even among isogenic cell lines. We then performed a high-throughput CRISPR screen to identify cell type-specific functional modifiers of ACE2 expression and cellular sensitivity to SARS-CoV-2 infection. 

In addition to identifying novel regulators of ACE2 expression, we also screened for genes that influenced the fitness of lung-derived cells during SARS-CoV-2 infection.  The top 4 hits from this screen all targeted the same type I interferon (IFN-I) signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. Surprisingly, despite the canonical antiviral properties of this pathway, we found that IFN-I signaling actually reduced the fitness of these cells. We subsequently found this to be due  to the induction of an antiproliferative response that restricted the expansion of cells surviving the initial peak of viral replication and cytopathic effect. Ongoing work is focused on dissecting the molecular mediators and in vivo significance of this response.