Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR
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.
Authors
- Claire Booth (ORCID: https://orcid.org/0000-0002-2626-5037)
- Roberto Nitsch (ORCID: https://orcid.org/0000-0001-8766-6267)
- Matthew H. Porteus (ORCID: https://orcid.org/0000-0002-3850-4648)
- Yiting Hu
- Alessia Cavazza (ORCID: https://orcid.org/0000-0002-4735-2121)
- Anne C.A. Mudde
- Adrian J. Thrasher (ORCID: https://orcid.org/0000-0002-6097-6115)
- Sridhar Selvaraj (ORCID: https://orcid.org/0000-0002-9736-8391)
- Marianna Romito
- Chinnu Rose Joseph (ORCID: https://orcid.org/0000-0003-4589-379X)
- Soraia P. Caetano-Silva (ORCID: https://orcid.org/0000-0001-7848-3266)
- Sandra Wimberger (ORCID: https://orcid.org/0000-0003-0541-7780)
- William N. Feist (ORCID: https://orcid.org/0000-0002-5870-4460)
- Nathan White (ORCID: https://orcid.org/0000-0002-1109-5100)
- Rajeev Rai (ORCID: https://orcid.org/0000-0003-4976-344X)
- Stina Svensson (ORCID: https://orcid.org/0000-0002-7843-789X)
- Giorgia Santilli (ORCID: https://orcid.org/0000-0003-1776-1984)
- Marcello Maresca (ORCID: https://orcid.org/0000-0003-0796-661X)
- Giandomenico Turchiano (ORCID: https://orcid.org/0000-0001-9980-6810)
- Grzegorz Sienski (ORCID: https://orcid.org/0000-0002-2730-7710)
- Panagiotis Antoniou (ORCID: https://orcid.org/0000-0002-0111-3897)
- Samuel Mark Pins
- John Alexander Chalk
Institutions
- California Institute for Regenerative Medicine (US)
- Great Ormond Street Hospital (GB)
- AstraZeneca (South Korea) (KR)
- AstraZeneca (Poland) (PL)
- AstraZeneca (Sweden) (SE)
- Institute of Infection and Immunity (CA)
- University College London (GB)
- Stanford University (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-11-03
- DOI
- https://doi.org/10.1038/s41467-025-65182-4
- Citations
- 1
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.81
Funders
- AstraZeneca
- Wellcome Trust
- European Federation of Pharmaceutical Industries and Associations
- University College London
- European Commission