Nature communicationsAug 26, 2026

Single-cell and live analyses show varied and organ-specific unintended effects and DNA rearrangements from CRISPR-Cas9

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

Updated Aug 29, 2026

Abstract

CRISPR-Cas9 may exhibit varied off-target activity across individual cells and different tissues.

Individual cells show unique off-target and translocation profiles, which are often overlooked in bulk analyses.
Sequence-independent features may influence Cas9 access and cleavage, favoring editing in areas with open chromatin and lower DNA methylation.
Different organs present distinct off-target spectra, varying patterns of DNA repair pathways, and differing tendencies for translocation.
Off-target activity is not uniform but varies significantly based on cellular context and tissue type.
These results highlight the need for precise single-cell analyses and organ-specific assessments in evaluating the safety of CRISPR therapies.

Simplified

CRISPR-Cas9 holds promise for treating genetic disease, but rare off-target mutations and structural variants remain as key safety concerns, especially at scales relevant to therapy. Here, we establish workflows to resolve Cas9 off-target activity in vitro at single-cell resolution and in vivo across different tissues. Using clonally expanded electroporated mouse embryos and embryonic stem cells, we reveal that individual cells exhibit unique off-target and translocation profiles, including events missed in bulk analyses. Integrating single-cell editing with chromatin accessibility, transcription, and DNA methylation measurements suggest that sequence-independent features modulate Cas9 access and cleavage, with preferential editing in regions characterized by open chromatin and lower methylation. In Cas9-inducible mouse models, editing analyses reveal organ-distinct off-target spectra, DNA repair pathway usage, indel patterns, and markedly varying translocation propensity between tissues. These findings demonstrate that off-target activity is heterogeneous across cells and context-dependent across organs, motivating sensitive single-cell analyses and organ-specific evaluation in preclinical development to more accurately assess risk and improve the safety of CRISPR-based genomic medicines.