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Updated: Jun 11, 2025
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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
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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.
Summary
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.
