CYP3A4and CYP3A5Expression is Regulated by CYP3A4*1Gin CRISPR/Cas9-Edited HepG2 Cells

Functional CYP3A4*1G(G>A, rs2242480) in cytochrome P450 3A4(CYP3A4) regulates the drug-metabolizing enzyme CYP3A4expression. The objective of this study was to investigate whether CYP3A4*1Gregulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4and CYP3A5in gene-edited human HepG2 cells. CYP3A4*1GGG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1GGA HepG2 cell line. In CYP3A4*1GGG, GA, and AA HepG2 cells, CYP3A4*1Gregulated expression of CYP3A4and CYP3A5mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4and CYP3A5was observed in CYP3A4*1GAA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1Gdecreased the induction level of CYP3A4and CYP3A5mRNA expression in CYP3A4*1GAA HepG2 cells. At the same time, CYP3A4*1Gdecreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1GGA HepG2 cells. In summary, we successfully constructed CYP3A4*1GGG and AA homozygous HepG2 cell models and found that CYP3A4*1Gregulates both basal and RIF-induced expression and enzyme activity of CYP3A4and CYP3A5in CRISPR/Cas9 CYP3A4*1GHepG2 cells.SIGNIFICANCE STATEMENTCytochrome P450 (CYP)3A4*1Gregulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4and CYP3A5. This study successfully established CYP3A4*1G(G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1Gregulates the basal and RIF-induced expression of CYP3A4and CYP3A5.