Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

Abstract

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knock-out, RNA interference knock-down, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks

“…CoV host factor discovery and validation is an active area of research, with multiple studies appearing in press and on preprint servers in recent months ( Baggen et al., 2020 ; Daniloski et al., 2020 ; Heaton et al., 2020 ; Hoffmann et al., 2020b ; Wang et al., 2020 ; Wei et al., 2020 ; Zhu et al., 2020b ). One group performed genome-wide CRISPR screens in the African green monkey kidney cell line VeroE6 and reported strong dependency on ACE2 and CTSL , consistent with our study ( Wei et al., 2020 ).…”

SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

“…Another study showed that entry of SARS-CoV-2 pseudoparticles into Calu-3 cells could be blocked using a clinically approved serine protease inhibitor (camostat mesylate) (Hoffmann et al, 2020b). In contrast, in most other cell lines (including the lung cell line A549), recent CRISPR-Cas9 screens have suggested that SARS-CoV-2 entry in cell lines appears to be mediated by endosomal cathepsins (Daniloski et al, 2020;Wang et al, 2020b;Wei et al, 2020a). These discrepancies between human lung cell lines underscore the importance of using relevant cells, such as human airway organoids (hAOs), to study SARS-CoV-2 entry.…”

Sex differences in viral entry protein expression and host transcript responses to SARS-CoV-2

“…In addition to reported immune response differences, we found vast differences in non-immune cells such as mitochondria functions, phagocytosis, and cholesterol biosynthesis, suggesting men and women have unique response trajectories after infection and latency. Both large-scale genome-wide CRISPR screenings confirmed that cholesterol synthesis is a key host response factor and targeting cholesterol synthesis using small molecules like 27-hydroxycholesterol could reduce SARS-CoV-2 infection in vitro [32][33][34] . Of note, independent of sex, younger people (<=60) have much more differentially expressed genes than older people (>60); independent of age, women tend to have more differentially expressed genes than men.…”