Cytochrome P450 family 1 subfamily A member 2 (CYP1A2) performs an indispensable role in the metabolism of both exogenous and endogenous substances. What is more, CYP1A2 functions in human diseases by regulating the homeostasis of cholesterol. Despite the emergence of gene-editing animal models, genetically humanized animals that overcome species differences for further exploring the role of CYP1A2 in drug metabolism and human diseases have not been constructed. In this study, we inserted human CYP1A2 cDNA into the rat Cyp1a2 gene by using CRISPR/CRISPR-associated protein 9 (Cas9) technology. The results showed that human CYP1A2 was successfully expressed in humanized rat liver, and there were no statistically significant differences of physiologic symptoms compared with wild-type (WT) rats. In vitro incubation results indicated the different inhibition of furafylline on CYP1A2 activity in human liver microsomes, humanized CYP1A2 (hCYP1A2) rat liver microsomes, and WT rat liver microsomes, with IC50 values of 7.1 μM, 36.5 μM, and 285.8 μM, respectively. Meanwhile, pharmacokinetic characteristics of clozapine were conducted, and the results suggested that in hCYP1A2 rats, clozapine tended to be metabolized into norclozapine. Both the in vitro and in vivo results demonstrated the different metabolic functions of CYP1A2 in humanized and WT rats. We successfully constructed a novel humanized CYP1A2 rat model using the CRISPR/Cas9 system, providing a powerful tool for better predicting CYP1A2-mediated drug metabolism and pharmacokinetics.

SIGNIFICANCE STATEMENT Human CYP1A2 takes active part in the biotransformation of both exogenous substances and endogenous substances. Meanwhile, it plays a regulatory role in human diseases, including hypercholesterolemia and hypertension. However, the results obtained from animal models cannot be directly applied to humans. This study successfully constructed a humanized CYP1A2 rat model by CRISPR/CRISPR-associated protein 9 technology, providing a powerful model for promoting drug metabolism research as well as exploring the role of CYP1A2 in human diseases.

This work was supported by grants from the National Natural Science Foundation of China [81773808 and 82274010], the Jointed PI Program from Shanghai Changning Maternity and Infant Health Hospital, the Innovative talent base for master and doctor of hypertensive disorder complicating pregnancy [RCJD2022S06], the East China Normal University (ECNU) Medicine and Health Joint Fund [2022JKXYD09004], and ECNU Construction Fund of Innovation and Entrepreneurship Laboratory.

The authors declare no conflicts of interest.

↵1J. Liu, J. Lu, and B.Y. contributed equally to this work.

dx.doi.org/10.1124/dmd.123.001500.