Fig. 2
A CRISPR knockout screen identifies IFITMs as blocking SAMHD1 degradation by Vpx upon IFN. α treatment. a Sorting strategy. 5 × 107 THP1 cells were treated with 1000 U/mL IFNα for 24 h and then incubated with 2.5 RT units of VLPs-Vpx pseudotyped with VSV-G for 16 h. Cells were harvested, incubated for 30 min with a viability dye, gently fixed with 1% PFA, permeabilized and stained for SAMHD1 as described before. SAMHD1 negative and SAMHD1 positive cells were sorted by flow cytometry on a BD FACS Aria-II using the indicated gates. The FSC/SSC gate allowed to sort out dead cells and debris, based on morphology. The doublet gates 1 and 2 allowed to remove cell doublets, based on height (H) and width (W) for the FSC and SSC parameters. The viability gate allowed to remove dying cells, that fluoresce in the DAPI channel and that exhibit aberant SAMHD1 staining. Cells were sorted on their levels of endogenous SAMHD1. The cutoff used for SAMHD1 negative cells is indicated in the red gate. 5 × 105 SAMHD1 negative, and 3 × 106 SAMHD1 positive cells were sorted to ensure sufficient library coverage. Two technical replicates were performed—one representative flow cytometry plot is shown. b Top 20 hits. sgRNA enrichment in SAMHD1 negative cells was determined after Illumina sequencing, and MAGeCK gene analysis was performed to take into account data from all 8 sgRNAs per gene and from two replicate experiments. The MAGeCK score (−log10) of the top 20 enriched genes is indicated on the X axis. Published IFNα induction data in THP1 cells [29] is shown on the Y axis (log scale). For some genes, expression was measured using multiple probes: in that case, we averaged the results. Two genes on our list, SAMHD1 and ATP8B4 (indicated with red asterisks), were absent from the dataset, and were arbitrarily set at 1. SAMHD1 is indicated with a blue square, positive controls, i.e. members of the IFN pathway, with orange triangles, and IFITM3 and IFITM1 with green diamonds. The entire dataset is presented in Additional file 1: Table S1