Identification of cell type specific ACE2 modifiers by CRISPR screening

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development. Author summaryThe amount of ACE2 on the surface of human cells is an important determinant of SARS-CoV-2 infection, but the molecular pathways that regulate ACE2 remain poorly understood. Identification of these pathways may clarify host factors involved in COVID-19 outcomes and offer targets for therapeutic development. ACE2-targeted therapies may furthermore be less susceptible than viral spike-targeted therapies to evasion by SARS-CoV-2 variants. To systematically identify regulators of human ACE2, we therefore performed high-throughput CRISPR screening for modifiers of ACE2 surface abundance in HuH7 liver-derived and Calu-3 lung-derived cell lines. Unexpectedly, aside from ACE2 itself, we identified distinct sets of ACE2 modifiers in either cell line. For a subset of ACE2 regulators, we validated their functional effect on ACE2, confirmed their relevance to SARS-CoV-2 infection, and clarified their level of regulation. Our findings demonstrate the important influence of cell type on investigations of SARS-CoV-2 infection and nominate candidate pathways for ACE2-targeted therapeutic development.