Fig 1: Genetic CYB5A and POR/CYB5A double-knockout in transduced HepaRG cells. (a) Location of CYB5A-targeting gRNAs relative to exon structure (gene chr18:74,250,846–74,292,016) indicating 4 translated exons as well as the binding region (black) for heme. The positions of two sgRNAs targeting exon 1 (CYB5#1) or exon 2 (CYB5#2) are indicated by arrows. (b) CYB5 mRNA and protein expression in transduced differentiated HepaRG cells quantified in cell lysates. Mean levels are shown relative to vector control (VC) set at 1 with SD bars (dark grey: VC, light grey: POR#1, white: POR#2). Results are means ± SD of three independent experiments. Statistical significance was assessed by unpaired t-test (c) Enzyme activities of seven CYP enzymes were determined simultaneously by cocktail LC–MS/MS assay in VC (dark grey), sgRNA POR#1 (light grey) and POR#2 (white) cells. Results are means ± SD of three independent experiments. Statistical significance was assessed by repeated measurements ANOVA with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).