Single-cell and in vivo profiling reveal heterogeneous and organ-specific CRISPR-Cas9 off-target and translocation outcomes
Year: 2026
Type: article
Abstract: 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. CRISPR can treat genetic disease, but unintended DNA changes remain a safety concern. Here, authors show that off-target editing and translocations vary widely between individual cells and organs, motivating the development of more sensitive and organ-specific safety assays for CRISPR therapies.
Source: Nature Communications
Authors: Alexandra Madsen, Niklas Selfjord, Marta Martinez-Lage, Anna-Lena Loyd, Gavin Kurgan, Mikaela Ståhlberg, Julia Lindgren, Julia Liz Touza, Leif Wigge, Mike Firth, Karl Nordström, Joseph Collin, Daniel Jachimowicz, Bastian Schiffthaler, Inken Dillmann, Panagiotis Antoniou, Aikaterini Emmanouilidi, Jonna Hellsten, Johan Forsström, Kerstin Magnell, Ashley Jacobi, Mark Behlke, Michelle Porritt, Katja Madeyski-Bengtson, Marcello Maresca, Pinar Akcakaya
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
Funder: AstraZeneca
OpenAlex ID: W7204264603