AviTag-seq unifies nucleotide-resolution maps of CRISPR off-targets and AAV vector integrations

Comprehensive safety assessment of gene-editing therapies requires quantifying both off-target cleavage and vector integration. However, current double-strand break (DSB)-dependent assays are fundamentally limited when evaluating nickase-based editors and are hindered by tag polarity constraints. Here, we present AviTag-seq, a platform repurposing AAV Inverted Terminal Repeats (ITRs) as universal capture tags. By exploiting the ITRs' single-stranded hairpin structure, AviTag-seq overcomes polarity issues, enabling high-sensitivity detection with a single primer pair, particularly in iPSCs. Crucially, it captures off-target events from prime and base editors that evade conventional detection. In vivo, AviTag-seq outperformed DISCOVER-Seq+ in profiling Pcsk9 off-targets in mouse liver while simultaneously mapping AAV integration sites. This dual profiling revealed that, unlike in vitro, AAV vectors in vivo preferentially integrate into active gene promoters, highlighting a specific genotoxic risk for liver-directed therapies. AviTag-seq thus offers a unified, regulatory-grade solution for evaluating diverse genome-editing modalities.