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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Comprehensive safety evaluation of gene-editing therapies necessitates accurate quantification of off-target cleavage and vector integration.
• Existing double-strand break (DSB)-dependent assays face limitations with nickase-based editors and tag polarity.
• There is a need for a unified assay to assess diverse gene-editing modalities.

Purpose of the Study:

• To develop and validate AviTag-seq, a novel platform for high-sensitivity detection of gene editing off-target events and vector integration.
• To overcome the limitations of current assays, including tag polarity constraints and applicability to nickase-based editors.
• To provide a unified, regulatory-grade solution for comprehensive safety assessment of gene-editing therapies.

Main Methods:

• Repurposing Adeno-Associated Virus (AAV) Inverted Terminal Repeats (ITRs) as universal capture tags.
• Exploiting the single-stranded hairpin structure of ITRs to overcome polarity issues.
• Utilizing a single primer pair for high-sensitivity detection, particularly in induced pluripotent stem cells (iPSCs).

Main Results:

• AviTag-seq demonstrated high-sensitivity detection of off-target events, including those from prime and base editors that evade conventional methods.
• In vivo studies in mouse liver showed AviTag-seq outperformed DISCOVER-Seq+ in profiling Pcsk9 off-targets and simultaneously mapped AAV integration sites.
• In vivo AAV vector integration was found to preferentially occur in active gene promoters, indicating a specific genotoxic risk for liver-directed therapies.

Conclusions:

• AviTag-seq offers a unified and sensitive platform for evaluating diverse genome-editing modalities, including nickase-based editors.
• The platform overcomes limitations of existing assays, providing a more comprehensive safety assessment.
• Findings highlight a specific genotoxic risk associated with in vivo AAV vector integration into gene promoters in liver-directed therapies.