Madsen, A.; Selfjord, N.; Martinez-Lage Garcia, M.; Loyd, A.-L.; Kurgan, G.; Stahlberg, M.; Lindgren, J.; Liz Touza, J.; Wigge, L.; Firth, M.; Nordstrom, K.; Collin, J.; Jachimowicz, D.; Schiffthaler, B.; Dillmann, I.; Antoniou, P.; Emmanouilidi, A.; Hellsten, J.; Forsstrom, J.; Magnell, K.; Jacobi, A.; Behlke, M.; Porritt, M.; Madeyski-Bengtson, K.; Maresca, M.; Akcakaya, P.

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. We established 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 suggested 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 revealed 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.