Long-read target enrichment sequencing for rAAV integration site analysis in engineered clones with targeted viral insertion.

Integration site analysis (ISA) plays a critical role in the genotoxicity assessment of recombinant adeno-associated viruses (rAAVs); however, ISA is challenged by the low integration rate, frequent vector genome rearrangements and the absence of well-characterized controls. Here, we generated 34 MCF10A-derived clones harboring recombinant adeno-associated viruses (rAAV) insertions at CRISPR-Cas9-induced double-strand breaks as cellular reference material and used these cell lines to evaluate long-read target enrichment sequencing (LR-TES) for rAAV ISA. LR-TES findings were complemented by droplet digital PCR quantification of vector copy number, long-range PCR analysis of on-target integration, and whole-genome sequencing on selected clones. Dilution experiments established a limit of detection of 1% under the tested conditions. LR-TES enabled genome-wide identification of vector-host junctions and structural characterization of integrated vector genomes, detecting targeted and non-targeted insertions, genomic deletions, and extensive vector restructuring across the clone panel. Only ∼29% of the integrated vector payload remained intact, while the observed truncations, rearrangements, or concatemers highlighted the structural complexity that ISA must resolve in rAAV settings. Together, the engineered clonal controls and optimized LR-TES provide reference material and a technical framework supporting rAAV ISA in safety evaluation, particularly when high structural resolution and specificity are needed to interpret predominant integration events.