dFLASH; dual FLuorescent transcription factor activity sensor for histone integrated live-cell reporting and high-content screening.

Live-cell transcription factor (TF) activity reporting is crucial for synthetic biology, drug discovery and functional genomics. Here we present dFLASH (dual FLuorescent transcription factor Activity Sensor for Histone-integrated live-cell reporting), a modular, genome-integrated TF sensor. dFLASH homogeneously and specifically detects endogenous Hypoxia Inducible Factor (HIF) and Progesterone Receptor (PGR) activities, as well as coactivator recruitment to synthetic TFs. The dFLASH system produces dual-color nuclear fluorescence, enabling normalized, dynamic, live-cell TF activity sensing with strong signal-to-noise ratios and robust screening performance (Z' = 0.61-0.74). We validate dFLASH for functional genomics and drug screening, demonstrating HIF regulation via CRISPRoff and application to whole-genome CRISPR KO screening. Additionally, we apply dFLASH for drug discovery, identifying HIF pathway modulators from a 1600-compound natural product library using high-content imaging. Together, this versatile platform provides a powerful tool for studying TF activity across diverse applications.