Identifying Essential Human Cell Factors for SARS-CoV-2 Infection
Summary
They carried out a genome-scale CRISPR loss-of-function screening to identify host factors necessary for SARS-CoV-2 viral infection of human alveolar epithelial cells to better understand host-virus genetic dependencies and discover potential therapeutic targets for COVID-19. The vacuolar ATPase proton pump, Retromer, and Commander complexes are a few examples of distinct pathways where top-ranked genes are grouped together. They use a variety of orthogonal techniques, including CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors, to validate these gene targets. They discover shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes using single-cell RNA sequencing. Additionally, they demonstrate that loss of RAB7A lowers viral entry by sequestering the ACE2 receptor inside cells, which is important given the role of the ACE2 receptor in the initial stages of viral entry. This work offers a genome-scale, quantitative resource of the effect of each host gene's loss on fitness/response to viral infection.
Fig.1 Graphical abstract. (Daniloski, 2021)
Research Criteria
To identify potential therapeutic targets for SARS-CoV-2, Daniloski et al. conduct a genome-wide CRISPR screening in human lung epithelial cells. They identified genes and pathways required for SARS-CoV-2 infection, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. Using single cell transcriptomics, they identified the upregulation of cholesterol biosynthesis as a common mechanism underlying viral resistance, in addition to ACE2 sequestration.
Sample Type
Human alveolar basal epithelial carcinoma cells (A549), human hepatocellular carcinoma (Huh7.5), human colorectal carcinoma (Caco-2), and lung adenocarcinoma (Calu-3) cells.
Result—Single Cell Sequencing Identifies Cholesterol Biosynthesis as a Common Mechanism Underlying Multiple Enriched Genes from the CRISPR Screening
To learn how individual genes identified in their loss-of-function screening prevent SARS-CoV-2 infection and whether host gene loss alters cell transcriptional programs, they combined pooled CRISPR perturbations of their top hit genes with single cell transcriptomics and proteomics readout using the expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) method. They discovered that removing six "perturbed" genes from the endosomal entry pathway — ATP6AP1, ATP6V1A, CCDC22, NPC1, PIK3C3, and RAB7A — resulted in similar gene expression signatures among upregulated differentially expressed genes. These six target gene mutations all resulted in the activation of pathways involved in lipid and cholesterol homeostasis. The researchers then measured cholesterol levels in cells after CRISPR perturbation and discovered that loss of these genes raises cholesterol by 10% to 50%, depending on the perturbation.
Fig.2 Single cell transcriptomics (ECCITE-seq) identifies shared target gene signatures for lipid and cholesterol. (Daniloski, 2021)
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Reference
- Daniloski, Z.; et al. Identification of required host factors for SARS-CoV-2 infection in human cells. Cell. 2021, 184(1): 92-105.e6.
