Multiplexed Sequencing-Based Host Cell Reactivation Assay Reveals New Dynamics In DNA Repair

Abstract

Defects in DNA repair are critical clinical biomarkers for therapeutic vulnerabilities in oncology. However, the characterization of DNA repair capacity remains hindered by the limited scalability of traditional functional assays. To overcome these constraints, we developed mmHCR-seq, a pioneering sequencing-based, massively multiplexed host-cell reactivation platform. By substituting conventional fluorescence signals with DNA barcodes and utilizing an RNA-sequencing readout from transiently transfected, lesion-bearing plasmids, this assay achieves unprecedented multiplexing capabilities. mmHCR-seq enables the parallel evaluation of both the efficiency and accuracy of major DNA repair pathways. The platform's single-nucleotide resolution uniquely reveals lesion-dependent kinetics and successfully captures transient base excision repair intermediates. Furthermore, by integrating mmHCR-seq with single-cell RNA-sequencing, we mapped functional repair capacities simultaneously in a pool of 19-cell lines at cellular resolution. This approach coupled repair phenotypes with transcriptomic states, elucidating complex pathway crosstalk and unmasking cell cycle dependencies without the need for cell synchronization. Rigorously validated for robustness and precision, mmHCR-seq provides a scalable, high-resolution framework for profiling the cellular repair landscape. Ultimately, this platform facilitates integration with large-scale multi-omics datasets, promising to significantly accelerate the discovery of mechanism-driven therapeutic targets, uncover novel cancer dependencies, and establish a powerful new paradigm for evaluating genomic stability and functional biomarkers in precision oncology.