Genome-wide CRISPR-Cas9 screen to identify SFV4 receptor

The goal of this study is to identify entry receptor for Semliki Forest virus (SFV, strain SFV4). For this, we used the human genome-wide Brunello CRISPR/Cas9 knockout library in a screen on human osteosarcoma cells (HOS, ATCC, CRL-1543) infected with fully replicative SFV4. While these cells are not a natural target for neurotropic SFV, they offer a highly SFV-permissive and easy to culture model needed for the screen. We performed the screening both with and without the janus kinase (JAK) inhibitor ruxolitinib as we hypothesized that ruxolitinib would limit the host cells capability to resist SFV replication and therefore sensitize the screen more towards factors associated with viral entry Generation of HOS-Cas9/BFP and transduction with lentiviral human genome-wide Brunello CRISPR-library as service by SciLifeLab CRISPR Functional Genomics unit (Karolinska Institutet, Stockholm, Sweden). 40 000 000 cells/sample were used in the screen to maintain full coverage of the library. For the screening cells were given either Ruxolitinib alone (10µM, Selleck Chemicals, #S5243) or Ruxolitinib and SFV4-d1-EGFP (MOI=50). Two days later the surviving cells were harvested sgRNA expression was analyzed with MAGeCK analysis by SciLifeLab CRISPR Functional Genomics unit.