Genome editing-based refinement of GPCR visualization in mice using the oxytocin receptor as a model.

G protein-coupled receptors (GPCRs) mediate diverse physiological functions and are major drug targets, yet their in vivo visualization remains challenging due to poor antigenicity. In this study, we integrate structural prediction, functional assay, and genome editing to systematically refine epitope-tagging strategies for GPCRs in mouse models. Using the oxytocin receptor (Oxtr) as a representative GPCR, we compare conventional triple-HA tagging with two recently developed high-sensitivity tags, Spaghetti Monster fluorescent protein (smFP) and the ALFA tag. Each tag is fused to the Oxtr C-terminus, and in vitro G protein activation assays show that all three variants retain ~ 70% of wild-type activity, consistent with structural predictions from AlphaFold 3. We then generate knock-in mice employing genome editing to evaluate in vivo performance. smFP markedly improves the signal-to-noise ratio in tissue staining, whereas the ALFA tag disrupts trafficking without enhancing sensitivity. Primary cultured neurons derived from smFP knock-in mice clearly visualize the distribution of Oxtr along dendrites down to their terminals at subcellular resolution. Collectively, the refined animal models advance GPCR biology and inform the design of future genome editing approaches.