Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Summary
Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 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 develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of 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 reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe 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 personalized treatment for ovarian cancer patients.
Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 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 develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of 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 reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe 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 personalized treatment for ovarian cancer patients.
Date Issued
2026-01-26
Date Acceptance
2025-12-02
Citation
Cell Reports: Methods, 2026, 6 (1)
ISSN
2667-2375
Publisher
Elsevier
Journal / Book Title
Cell Reports: Methods
Volume
6
Issue
1
Copyright Statement
© 2025 The Authors. Published by Elsevier Inc. User License: Creative Commons Attribution (CC BY 4.0)
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Publication Status
Published
Article Number
ARTN 101270
Date Publish Online
2025-12-30