Fig. 2. Genome-wide CRISPR-Cas9 knockout screening of warfarin-resistant VKR.

a Schematic diagram of genome-wide loss-of-function screening using Brunello lentiviral library with FIXgla-Fas/HEK293 reporter cells. This figure was created with BioRender.com. b Scheme of VKD carboxylation of reporter protein FIXgla-Fas by the vitamin K redox cycle. Apoptosis of reporter cell occurs only when the reporter protein FIXgla-Fas is carboxylated. c Scatterplot of sgRNA enrichment after VKD apoptotic functional screening. Red dots, Fas-associated apoptosis pathway associated proteins; green dots, calcium-dependent proteins; purple dots, GGCX. d Cell-based validation of the candidate genes for vitamin K reduction in the presence of warfarin. Lentivirus containing Cas9 and selected sgRNA were transduced into FIXgla-Met.Luc/HEK293 reporter cells. After puromycin selection for 7 days, survival cells were incubated with 11 µM vitamin K and 5 µM warfarin for 24 h. Carboxylation efficiency of the reporter protein FIXgla-Met.Luc was determined by luminescence ELISA. VKD carboxylation efficiency of non-targeting sgRNA transfected cells was normalized to 100%. e Immunoblotting of HEK293 cells and these cells with their fsp1 gene knocked out (FSP1 KO). Top panel: probed by anti-FSP1 antibody; bottom panel (loading control): probed by anti-GAPDH antibody. Full-length FSP1 is indicated by an arrowhead. f Effect of FSP1 knockout on VKR activity in HEK293 cells. FSP1 was knocked out (-FSP1) from FIXgla-PC/HEK293 or it was re-introduced back into the knockout cells (+FSP1) for VKR activity assay. The carboxylation activity of FSP1 transfected cells (+FSP1) was normalized to 100%. Bottom: Immunoblotting of FSP1 in the corresponding cells using anti-FSP1 as the primary antibody. g HPLC-based conventional VKR in vitro activity assay to determine the reduction of vitamin K to KH₂. Control: reaction buffer without cell lysate; HEK293, cell lysate of HEK293; HEK293 + FSP1: cell lysate of HEK293 overexpressing FSP1. A same number of HEK293 and HEK293 + FSP1 cells were used for the activity assay. h Warfarin inhibition of FSP1 reducing vitamin K to KH₂ by in vitro activity assay as described above (Fig. 2g). Final concentration of warfarin in the reaction mixture was 100 μM. Data are presented as mean ± SD of three independent experiments (n = 3) in Fig. 2d, f, h. Similar results were observed at least twice as shown in Fig. 2e, f.