Long-term in vivo CRISPR/Cas9 gene editing in liver triggers host immunity and clonal mutations in DNA damage response genes beyond off-target effects

CRISPR-Cas9 genome editing holds therapeutic promise but raises safety concerns beyond off-target effects, including p53 pathway activation and immunogenicity. The long-term physiological consequences of these stressors in vivo remain undefined. Here, in two mouse models of AAV8-mediated, long-term liver editing (Pcsk9, Angptl3), a 10-month follow-up reveals a potent adaptive immune response, leading to chronic hepatic inflammation and fibrosis. Integrated analysis further demonstrates suppression of apoptotic pathways, clonal enrichment of nonsynonymous mutations in DNA damage response genes, and increased chromosomal translocations. Our study uncovers a pro-tumorigenic milieu shaped by immunity and genomic instability, highlighting the need to expand the safety assessment of in vivo gene editing.