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<p>Genome editing by CRISPR-Cas9-nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted <i>locus</i>. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assess editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting <i>HBG1/2p</i>, scSNP-DNA-seq reveals that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.</p>

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Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity and reveal its mitigation by palbociclib and long-term engraftment

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月10日

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  • Julian Boutin,
  • Sabrina Fayet,
  • Victor Marin,
  • Camille Bergès,
  • Maude Riandière,
  • Jérôme Toutain,
  • Isabelle Lamrissi-Garcia,
  • Chloé Thibault,
  • David Cappellen,
  • Sandrine Dabernat,
  • Arthur Poulet,
  • Maëla Francillette,
  • Nathalie Droin,
  • Christelle Debeissat,
  • Philippe Brunet de la Grange,
  • François Moreau-Gaudry,
  • Aurélie Bedel

摘要

Genome editing by CRISPR-Cas9-nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted locus. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assess editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting HBG1/2p, scSNP-DNA-seq reveals that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.