Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

Nathan White, John Alexander Chalk, Yi-Ting Hu, Samuel Mark Pins, Chinnu Rose Joseph, Panagiotis Antoniou, Sandra Wimberger, Stina Svensson, Soraia Patricia Caetano-Silva, Anne Charlotte Adriane Mudde, Rajeev Rai, Sridhar Selvaraj, William Nelson Feist, Marianna Romito, Grzegorz Sienski, Roberto Nitsch, Claire Booth, Giorgia Santilli, Alessia Cavazza, Matthew Hebden Porteus, Marcello Maresca, Adrian James Thrasher and Giandomenico Turchiano ()
Additional contact information
Nathan White: University College London
John Alexander Chalk: University College London
Yi-Ting Hu: University College London
Samuel Mark Pins: University College London
Chinnu Rose Joseph: AstraZeneca
Panagiotis Antoniou: AstraZeneca
Sandra Wimberger: AstraZeneca
Stina Svensson: University College London
Soraia Patricia Caetano-Silva: University College London
Anne Charlotte Adriane Mudde: University College London
Rajeev Rai: University College London
Sridhar Selvaraj: Stanford University
William Nelson Feist: Stanford University
Marianna Romito: University College London
Grzegorz Sienski: AstraZeneca
Roberto Nitsch: AstraZeneca
Claire Booth: University College London
Giorgia Santilli: University College London
Alessia Cavazza: University College London
Matthew Hebden Porteus: Stanford University
Marcello Maresca: AstraZeneca
Adrian James Thrasher: University College London
Giandomenico Turchiano: University College London

Nature Communications, 2025, vol. 16, issue 1, 1-20

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-65182-4

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