Nathan White, John Alexander Chalk, Yiting Hu, Samuel Mark Pins, Chinnu Rose Joseph, Panagiotis Antoniou, Sandra Wimberger, Stina Svensson, Soraia P. Caetano-Silva, Anne C.A. Mudde, Rajeev Rai, Sridhar Selvaraj, William N. Feist, Marianna Romito, Grzegorz Sienski, Roberto Nitsch, Claire Booth, Giorgia Santilli, Alessia Cavazza, Matthew H. Porteus, Marcello Maresca, Adrian J. Thrasher, Giandomenico Turchiano
DNA repair mechanisms in human primary cells, including error-free repair, and, recurrent nuclease cleavage events, remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR), an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs, small indels, large deletions, and other aberrations in absolute terms in clinically relevant edited cells, including HSPCs, iPSCs, and T-cells. By quantifying up to 90% of loci with unresolved DSBs, CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore, we accurately quantify DNA repair precision, revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics, paving the way for mechanistic studies to advance gene therapy, designer editors, and small molecule discovery.