Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionised the treatment of high-grade serous ovarian carcinoma (HGSC), particularly in homologous recombination-deficient tumours. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within three years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood as current experimental methodologies lack resolution and throughput. To address this, we developed an intramolecular FRET-based biosensor by CRISPR-Cas9 dual-labelling endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells. This biosensor enables real-time, single-cell analysis of PARP trapping dynamics. Using fluorescence lifetime imaging microscopy (FLIM), we revealed dose-dependent PARP trapping, differentiated the trapping efficiencies of four clinically approved PARPi and observed reduced trapping in PARPi-resistant models in vitro and in vivo. This biosensor provides critical insights into PARPi resistance mechanisms, with implications for developing more effective therapies and advancing personalised treatment for ovarian cancer patients.