# Development of a Novel Method to Detect AAV Vector Integration

## TL;DR

This paper introduces a new PCR-free method using CRISPR-Cas9 and long-read sequencing to accurately detect AAV integration sites in gene therapy.

## Contribution

A novel PCR-free workflow combining CRISPR-Cas9 and long-read sequencing for unbiased AAV integration detection.

## Key findings

- The CRISPR-Cas9-based workflow preserves native AAV integration states and detects integration junctions without PCR bias.
- The method showed strong consistency with probe hybridization and Illumina sequencing in identifying AAV integration sites.
- AAV integration junctions were confirmed by PCR, validating the accuracy of the new approach.

## Abstract

AAV integration has become an important safety consideration in gene therapy. However, accurately determining integration sites remains challenging due to biases introduced by library preparation methods, sequencing technologies, and bioinformatic pipelines. In this study, we developed a PCR-free amplification based on a CRISPR-Cas9 cleavage strategy for AAV DNA that overcomes the limitations of PCR amplification imposed by the ITR structure. When combined with long-read nanopore sequencing, this CRISPR-Cas9-based workflow preserves native AAV integration states and enables unbiased detection of integration junctions. We used AAV-transduced HeLa single-cell clones to evaluate the performance of this approach. To confirm integration site identification, AAV integration junctions were also detected using a probe hybridization capture strategy followed by Illumina short-read sequencing. Integration junctions identified by both methods were further confirmed by PCR. The results showed strong consistency between the two approaches in accurately identifying AAV integration sites in each clone. Overall, these findings demonstrate that the CRISPR-Cas9-enabled, PCR-free long-read sequencing workflow provides a promising tool for characterizing AAV integration events.