Pré-Publication, Document De Travail Année : 2025

Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells

Julian Boutin
Sabrina Fayet
  • Fonction : Auteur
Victor Marin
Camille Bergès
  • Fonction : Auteur
Maude Riandière
  • Fonction : Auteur
Jérôme Toutain
Isabelle Lamrissi-Garcia
  • Fonction : Auteur
Chloé Thibault
  • Fonction : Auteur
David Cappellen
Sandrine Dabernat
Arthur Poulet
Maëla Francillette
  • Fonction : Auteur
Nathalie Droin
  • Fonction : Auteur
François Moreau-Gaudry
Aurélie Bedel

Résumé

Genome editing by CRISPR-Cas9 is promising for genetic disease and cancer gene therapy. However, safety concerns are still present, particularly the ON-target genotoxicity for protocols using nucleases. Quality control of edited cells before and after graft is mandatory, especially to assay megabase-scale genomic rearrangements induced at the targeted locus . These unintended events are fortunately rare but potentially deleterious. Classical PCR-based bulk approaches do not detect them or underestimate their frequency. Single-cell approaches are promising but RNA sequencing only estimates copy number variation of the genome. Here, we propose single-cell DNA sequencing to accurately evaluate and monitor CRISPR-mediated genotoxicity in primary cells (human fibroblasts and hematopoietic stem/progenitor cells). We designed a homemade panel using single nucleotide polymorphisms to detect, map and characterize induced-losses of heterozygosity (terminal, interstitial, copy-loss and copy-neutral). This innovative approach revealed intense genotoxicity linked to the double strand break. The risk was associated with DNA repair modulators in particular DNA-PKcs inhibitor AZD7648. Importantly, the CDK4/6 inhibitor palbociclib prevented these rearrangements in hematopoietic stem/progenitor cells. This work strongly suggests that single-cell DNA sequencing should be routinely implemented in clinical applications before CRISPR-edited cell infusions. Graphical abstract

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Dates et versions

hal-05159818 , version 1 (12-07-2025)

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Julian Boutin, Sabrina Fayet, Victor Marin, Camille Bergès, Maude Riandière, et al.. Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells. 2025. ⟨hal-05159818⟩
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