Generation of Cre/LoxP-mediated extracellular TurboID knock-in rats with CRISPR/Cas9 system.

The plasma membrane plays a central role in regulating signal transduction from the extracellular environment to the intracellular space and represents a major site of drug targeting. Proximity-dependent biotinylation with biotin ligases such as BioID and its derivatives, including TurboID and AirID, enables identification of novel protein‒protein interactions by fusion to a target protein. In vivo application of biotin ligases for cell membrane analysis has been attempted; however, methods for expressing biotin ligases on the target cell membrane remain largely limited to viral vector delivery. In this study, we inserted a Cre-dependent TurboID expression cassette into the rat genome to express TurboID on the cell surface via the CRISPR/Cas9 system. We generated TurboID knock-in (KI) rats that express TurboID on the cell surface, enabling biotinylation of extracellular proteins. The TurboID KI rats thus provide a valuable model for in vivo analysis of cell-surface molecules and may facilitate identification of novel drug targets or antigens.