Related Experiment Video
Updated: Jun 11, 2025
Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
AAV vector-derived elements integrate into Cas9-generated double-strand breaks and disrupt gene transcription
Hannah O Bazick1, Hanqian Mao2, Jesse K Niehaus1
1UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Abstract:
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 3 additional AAV vectors containing S. aureus Cas9 in vitro and in vivo, and 25 vectors containing N. meningitidis 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 method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including AAV integration and unresolved double-strand breaks. 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 poly(A) 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.
Insights
New adeno-associated virus (AAV) gene therapies for Angelman syndrome using single-target Cas9 were less effective than multi-target vectors. AAV integration was the most common editing event, influencing gene expression.
Area of Science:
- Gene Therapy
- Molecular Biology
- Neuroscience
Background:
- Previous adeno-associated virus (AAV) Cas9 gene therapy for Angelman syndrome integrated into the genome, causing premature termination of Ube3a-ATS.
- Angelman syndrome is a neurodevelopmental disorder caused by the loss of function of the UBE3A gene.
Purpose of the Study:
- To assess the efficacy of novel AAV vectors with Staphylococcus aureus Cas9 and Neisseria meningitidis Cas9 for Angelman syndrome.
- To compare the effectiveness of single-target versus multi-target guide RNA (gRNA) vectors in reducing Ube3a-ATS expression.
- To characterize AAV integration and other editing events at target sites within Ube3a-ATS.
Main Methods:
- In vitro and in vivo assessment of 3 AAV vectors with S. aureus Cas9 and 25 AAV vectors with N. meningitidis Cas9, targeting single sites in Ube3a-ATS.
- Development of an anchored multiplex PCR sequencing method and analysis pipeline to quantify editing events, including AAV integration and double-strand breaks.
- Use of a Ube3a-ATS minigene reporter to evaluate the impact of insertions/deletions (indels) and vector-derived elements on transcription.
Main Results:
- Single-target gRNA vectors were less effective than multi-target vectors in reducing Ube3a-ATS expression in neurons.
- AAV integration was the predominant editing event (67%-89%) at single target sites, exceeding insertions and deletions (indels).
- Vector-derived elements (poly(A) and reverse promoter) reduced downstream transcription by up to 50%, while common indels did not.
Conclusions:
- Multi-target gRNA strategies are more effective for reducing Ube3a-ATS expression compared to single-target approaches.
- AAV integration is the primary editing outcome, and its gene-trapping potential is influenced by integrated vector elements.
- The number of target sites and specific vector-derived elements impact the frequency and consequences of AAV integration events.
