High levels of AAV vector integration into CRISPR-induced DNA breaks

TLDR

AAV vectors show promise in preclinical models, yet the genomic impact of delivering CRISPR‑Cas nucleases via AAV remains under investigation. The authors engineered a miniature AAV carrying a 465‑bp lambda DNA cassette (AAV‑λ465) to enable full‑genome sequencing of integrated vectors. They found that AAV integrates into Cas9‑induced double‑strand breaks at rates up to 47% in neurons, brain, muscle, and cochlea, with both full‑length and fragmented genomes localized to the target site, indicating that AAV integration is a common outcome of AAV‑mediated genome editing.

Abstract

Abstract Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.