Reversible in vivo regulation of drug metabolizing enzyme CYP1A2 activity through a dTAG knock-in strategy

Abstract Drug-metabolizing enzymes determine therapeutic exposure, efficacy and toxicity, but defining their isoform-specific functions in vivo remains challenging. Cytochrome P450 enzymes (P450s) are central to drug metabolism and pharmacokinetics (DMPK) and mediate the phase I metabolism of ∼75% of all marketed drugs. However, conventional knockout models can induce develop-mental and compensatory adaptations, and selective inhibitors are unavailable for many P450 isoforms. Here, we report the use of an inducible chemical-genetic platform for acute and specific degradation of the endogenous P450 enzyme Cyp1a2 in mice. Using CRISPR-Cas9-mediated knock-in editing, we introduced an FKBP12 F36V degron into the endogenous Cyp1a2 locus to generate Cyp1a2 dTAG mice. Treatment with the dTAG degrader dTAG-13 recruited an E3 ubiquitin ligase to CYP1A2 dTAG , resulting in rapid and reversible proteasomal depletion of CYP1A2 dTAG in vivo. Temporally controlled CYP1A2 dTAG loss altered caffeine pharmacokinetics as expected, validating this model as a functional tool for DMPK studies. By enabling reversible suppression of drug-metabolizing enzymes without permanent deletion or chronic inhibitor exposure, this work establishes targeted protein degradation as a broadly adaptable strategy for studying drug metabolism in vivo and provides a foundation for extending inducible DMPK control to other P450s, conjugating enzymes and transporters.