Abstract
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.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (Grant Nos. 2019YFA0110803, 2019YFA0110204, and 2021YFA1100900), the National Natural Science Foundation of China (Grant Nos. 92568302, 82570286, 82402188, 81870149, 82070115, 81890990 and 81730006), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (Grant Nos. 2024-I2M-ZH-015, 2024-I2M-3-018, 2023-I2M-2-007, 2022-I2M-2-003, 2022-I2M-2-001, 2021-I2M-1-041, 2021-I2M-1-040, and 2021-I2M-1-001), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (Grant No. 2020-PT310-011), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (Grant No. TSBICIP-KJGG-017), the CAMS Fundamental Research Funds for Central Research Institutes (Grant No. 3332021093), the Tianjin Natural Science Foundation Project (Grant No. S25YB0754), the Haihe Laboratory of Cell Ecosystem Innovation Fund (Grant Nos. 24HHXBSS00005 and HH22KYZX0022), the China Foundation For Youth Entrepreneurship and Employment-Incaier Public Welfare Fund (HH25KYHX0009), the Fundamental Research Funds for the Central Universities (Grant No. 3332024074) and Postdoctoral Fellowship Program of CPSF (Grant Nos. GZC20240154).
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Li, JX., Zhang, SM., Ma, XY. et al. AviTag-seq unifies nucleotide-resolution maps of CRISPR off-targets and AAV vector integrations. Commun Biol (2026). https://doi.org/10.1038/s42003-026-10298-6
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DOI: https://doi.org/10.1038/s42003-026-10298-6
