SARS-Cov2 CRISPR genome wide

Despite recent advances in the development of vaccines and antivirals, COVID-19 remains a thread to public health. In order to reveal dependency factors promoting SARS-CoV-2 infection, we conducted two genome-wide CRISPR knock-out screens in the human lung epithelial cell lines A549 and Calu-3. A meta-analysis including our data and the data of four published CRISPR screens performed in different cells identified 28 potential dependency factors including the ACE2 receptor of SARS-CoV-2. We validated these hits by knock-down screening in A549 cells, cytotoxicity assays and expression analysis of the selected genes in relevant tissues. In these experiments we identified RAB9A, a GTPase involved in retrograde trafficking from late endosomes as a SARS-CoV-2 dependency factor. RAB9A supported SARS-CoV-2 infection in various cell types. Pseudovirus experiments revealed involvement of RAB9A in entry that was mitigated upon ectopic expression of the host protease TMPRSS2, suggesting an involvement in the endocytic entry pathway. By dissecting the different steps of SARS-CoV-2 entry, we excluded potential effects of RAB9A on virus binding and were able to show an increased rate of virus internalization followed by retention of virus in lysosomes of RAB9A-depleted cells. The mechanism by which RAB9A promotes SARS-CoV-2 entry seems furthermore to be supported by the RAB9A-specific GDP-GTP exchange factor DENND2D, indicating that RAB9A activity is required for its proviral function. In summary, our data show that RAB9A supports later steps of viral entry, such as fusion, endosomal exit or uncoating of the virion.