Massively Multiplexed Sequencing-Based Host Cell Reactivation Assay Profiles DNA Repair at Single-Nucleotide and -Cell Resolution [scRNA]

However, the characterization of DNA repair in cancer remains bottlenecked by a lack of reliable genomic predictors and the low throughput of existing functional assays. Here, we present mmHCR-seq, a sequencing-based and massively multiplexed Host Cell Reactivation assay that enables parallel quantification of functional repair efficiency and accuracy across all major repair pathways via RNA-seq readout.

We demonstrate that mmHCR-seq can detect deficiencies and measure kinetics of nine DNA repair pathways simultaneously. The single-nucleotide resolution revealed lesion-dependent kinetics for base excision repair intermediates. By integrating mmHCR-seq with single-cell RNA-seq, we mapped repair capacity in a 19-cell line pool at cellular resolution and coupled repair phenotypes to expression states.