Genome-wide CRISPR Knockout Library Screening

Cells were either transfected with Cas9–2A-GFP (pX458; Addgene #48138) containing a guide RNA from the TKOv3 library (Addgene #125517) or co-transfected with Cas9–2A-GFP (pX458; Addgene #48138) together with pLV-U6g-PPB encoding a guide RNA from the LUMC/Sigma-Aldrich sgRNA library using lipofectamine 2000 (Invitrogen) (sgRNAs are listed in Supplementary Table 2 and plasmids in Supplementary Table 3). Cells were FACS sorted on BFP/GFP and plated at low density after which individual clones were isolated. Alternatively, cells were selected with puromycin (1 μg/mL) for 3 days and seeded at low density after which individual clones were isolated. Isolated knockout clones were verified by western blot analysis and/or Sanger sequencing previously described (Primers are listed in as Supplementary Table 4) ^{6 (link)}.