CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells

Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regu-lates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CY-P3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-asso-ciated protein 9 (CRISPR/Cas9) single nucleotide polymorphism tech-nology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele -de-pendent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G de-creased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CY-P3A4*1G GA HepG2 cells. In summary, we successfully constructed CY-P3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and en-zyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells.SIGNIFICANCE STATEMENTCytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and 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*1G regu-lates the basal and RIF-induced expression of CYP3A4 and CYP3A5.