AAV vector-derived elements integrate into Cas9-generated double strand breaks and disrupt gene transcription

We previously developed an adeno-associated virus (AAV) Cas9 gene therapy for Angelman syndrome that integrated into the genome and prematurely terminated Ube3a-ATS. Here, we assessed the performance of three additional AAV vectors containing S. aureus Cas9 in vitro and in vivo, and twenty-five vectors containing N. meningococcus Cas9 in vitro, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed an anchored multiplex PCR sequencing (AMP-seq) method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including AAV integration and unresolved double-stranded breaks (DSBs). We found that integration of AAV was the most frequent editing event (67-89% of all edits) at three different single target sites, surpassing insertions and deletions (indels). None of the most frequently observed indels were capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements-the polyA and reverse promoter-reduced downstream transcription by up to 50%. Our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.