Single-Cell Sequencing Maps CRISPR-Cas9 Chromosomal Damage

Double-strand breaks induced by CRISPR-Cas9 nuclease can trigger megabase-scale chromosomal rearrangements, raising safety concerns for gene therapy applications. The research team combined single-cell DNA sequencing focused on single nucleotide polymorphisms (scSNP-DNAseq) with micronuclei detection and loss-of-heterozygosity assays to comprehensively monitor on-target genotoxicity.

Using this approach in haematopoietic stem cells, they detected both interstitial and terminal chromosomal rearrangements affecting up to 9% of edited cells when targeting the HBG1/2 promoters. The DNA-PKcs inhibitor AZD7648, proposed to enhance homology-directed repair, increased genotoxicity three-fold. Conversely, transient cell cycle arrest using palbociclib – a CDK4/6 inhibitor – reduced megabase-scale rearrangements seven-fold without compromising editing efficiency or engraftment capacity. Importantly, analysis of bone marrow from transplanted mice revealed complete disappearance of chromosomally rearranged cells after three months in vivo, even following AZD7648 exposure.

The findings demonstrate that scSNP-DNAseq enables precise detection and mapping of CRISPR-induced chromosomal instability, whilst cell cycle modulation offers a strategy to mitigate genotoxic risk in clinical gene therapy protocols.

The study was led by François Moreau-Gaudry and Aurélie Bedel from University of Bordeaux, France. It was published in Nature Communications on 10 January 2026.