Genetically Encoded Cell Fate Reporter System for Multiplex Single-Cell Detection of DNA and Mitochondrial Damage

Predicting the mechanism of action of bioactive compounds and toxicants is of great importance in drug discovery, as well as toxicology testing of environmental toxicants. Many reporter cell lines were created for generating phenotypic data in assigning the biological impact of large compound libraries. A major requirement of a phenotypic screen is the reporter cell lines for appropriate biomarkers with a good predictive value. Here, we have developed a stable cell expressing TagBFP2-53BP1 as a real-time sensor for DNA damage at the single-cell level that marks 53BP1 foci formation under physiological conditions, as well as toxicant-induced DNA damage. The cells were further engineered to report mitochondrial permeabilization using Smac-RFP, providing a broader application potential for this cellular model. Once the cells are further expressed with the autophagy marker EGFP-LC3, it is possible to image distinct cell fates such as autophagy, mitochondrial permeabilization, and DNA damage at the single-cell level. Using this system, we demonstrate that early cell death induced by telomerase inhibitors involves double-strand breaks and mitochondrial permeabilization. The model also enabled systematic profiling of several toxicants based on clearly noticeable cell fates. The approach of using live single-cell sensors for multiple distinct and diverse phenotypes offers great advantages over independent single-parameter-based assays, with the additional benefit of extracting heterogeneous cell responses over time, revealing more insight into the toxicity and mechanism of action for predictive applications.