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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月3日

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  • 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,
  • Giandomenico Turchiano

摘要

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.