idTAG enables programmable and physiologically faithful control of endogenous protein degradation

Abstract

Rapid and precise depletion of endogenous proteins is essential for dissecting gene function, yet existing degron technologies often require direct modification of endogenous coding sequences, which can compromise protein expression and limit their applicability. Here, we develop idTAG, an inducible degron platform that decouples protein expression from protein degradation by integrating Tet-On 3G-mediated transcriptional control with dTAG-mediated degradation. Systematic characterization reveals that conventional degron knock-in frequently causes unintended reduction of target protein expression at the mRNA level, representing an underappreciated limitation of current approaches. In contrast, idTAG enables efficient generation of degron cell lines while maintaining physiological protein abundance and allows programmable control of degradation kinetics through independent regulation of protein synthesis and degradation. Applying idTAG to RNA-binding proteins, we demonstrate that acute depletion of FUS has minimal effects on global RNA polymerase II transcription, whereas acute loss of SRSF5 selectively disrupts transcription termination at a subset of genes. Thus, idTAG provides a versatile platform for physiological and temporally controlled interrogation of endogenous protein functions. Highlights Endogenous degron knock-in frequently reduces target mRNA and protein abundance idTAG preserves physiological protein levels, enabling efficient generation of degron cell lines Independent control of mRNA expression and protein degradation of idTAG tunes depletion kinetics FUS loss has little effect on transcription, whereas SRSF5 loss selectively impairs transcription termination Graphical abstract