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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
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High levels of AAV vector integration into CRISPR-induced DNA breaks
Killian S Hanlon1,2, Benjamin P Kleinstiver3,4,5, Sara P Garcia5,6,7
1Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA.
Nature Communications
|October 2, 2019
Summary
Adeno-associated virus (AAV) integration into CRISPR-Cas9 induced breaks is common in gene editing applications. This study found high AAV integration rates, highlighting its significance for therapeutic genome editing safety.
Area of Science:
- Molecular Biology
- Gene Therapy
- Genomics
Background:
- Adeno-associated virus (AAV) vectors are promising for gene therapy.
- Genomic consequences of AAV vectors encoding CRISPR-Cas nucleases require examination.
Purpose of the Study:
- To investigate AAV integration frequency and characteristics at CRISPR-Cas9 induced double-strand breaks (DSBs).
- To characterize the integration profile of engineered AAV vectors.
Main Methods:
- Assessed AAV integration into Cas9-induced DSBs in cultured murine neurons, mouse brain, muscle, and cochlea.
- Performed genome-wide AAV mapping in mouse brain.
- Engineered a miniature AAV (AAV-λ465) for detailed integration analysis.
Main Results:
- Observed high AAV integration rates (up to 47%) at Cas9 target sites in various tissues.
- Genome-wide mapping showed AAV integration primarily at CRISPR/Cas9 target sites.
- Sequencing of AAV-λ465 revealed both full-length and fragmented AAV genomes at on-target sites.
Conclusions:
- AAV integration is a common outcome in genome editing applications utilizing AAV vectors.
- Understanding AAV integration is crucial for the safety and efficacy of gene editing therapies.
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