CRISPR–Cas9 pooled screening to identify regulators of GPI biosynthesis. (A) Graphic depiction of the GPI biosynthesis pathway in mammalian cells. Genes in blue were significant by MAGeCK analysis, genes in red were complexes of GPI transamidases, and genes in black showed no enrichment. (B) Validation of the screening results via the expression of related genes in KO cells. Cells were stained with T5 mAb and analyzed by FACS. (C and (D) Flow cytometry analysis of the KO of ARV1, CLPTM1L, and SRD5A3 in HEK293 WT cells. Cells were stained with anti-CD59 and quantified. Relative normalized geometric MFI in KO cells is compared with WT cells and displayed as the mean ± SD from three independent experiments (one-way ANOVA followed by Dunnett’s multiple comparisons test).