Citation
- Authors: Rebendenne A. et al.
- Year: 2022
- Journal: Nat Genet 54 1090-1102
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
The pCSI-spikeRBD expression vector was transfected in HEK293T cells using the PEIpro transfection reagent. Cells were washed 6 h after transfection and grown for an additional 72–96 h in serum-free Optipro medium (Invitrogen) supplemented with glutamine and non-essential amino acids. Supernatant was harvested, filtered and concentrated 100-fold on 10 kDa-cutoff Amicon Ultra-15 concentrators. Samples were aliquoted and stored at −20 °C until further use. The spike RBD-mFc validation is presented in Supplementary Fig. 2.
Abstract
CRISPR knockout (KO) screens have identified host factors regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Here, we conducted a meta-analysis of these screens, which showed a high level of cell-type specificity of the identified hits, highlighting the necessity of additional models to uncover the full landscape of host factors. Thus, we performed genome-wide KO and activation screens in Calu-3 lung cells and KO screens in Caco-2 colorectal cells, followed by secondary screens in four human cell lines. This revealed host-dependency factors, including AP1G1 adaptin and ATP8B1 flippase, as well as inhibitors, including mucins. Interestingly, some of the identified genes also modulate Middle East respiratory syndrome coronavirus (MERS-CoV) and seasonal human coronavirus (HCoV) (HCoV-NL63 and HCoV-229E) replication. Moreover, most genes had an impact on viral entry, with AP1G1 likely regulating TMPRSS2 activity at the plasma membrane. These results demonstrate the value of multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential targets for therapeutic interventions.