Nature communicationsJan 10, 2026

Detailed single-cell analysis of CRISPR DNA damage and its reduction by palbociclib during long-term cell transplantation

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

A single-cell multiplex assay mapped on-target CRISPR genotoxicity and suggested palbociclib and long-term engraftment can reduce detectable rearrangement risk.

Evidence

This platform and preclinical primary-cell study combined scSNP-DNAseq, micronuclei assays, LOH reporters, hematopoietic stem cells, and long-term xenografts to track CRISPR-associated chromosomal instability.

Caveat

The mitigation findings come from assay systems and xenografts rather than clinical cell-infusion outcomes.

Simplified

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.

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Detailed single-cell analysis of CRISPR DNA damage and its reduction by palbociclib during long-term cell transplantation

Abstract

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