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Nat Commun. 2019 Sep 30;10(1):4439. doi: 10.1038/s41467-019-12449-2.

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

Author information

1
Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA.
2
Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, 02129, USA.
3
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
4
Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
5
Department of Pathology, Harvard Medical School, Boston, MA, USA.
6
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, 02129, USA.
7
Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
8
Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA.
9
Department of Gynecology, Obstetrics and Gynecologic Oncology, Division of Gynecologic Oncology, Poznań University of Medical Sciences, 60-535, Poznań, Poland.
10
Institute of Molecular and Clinical Ophthalmology Basel, 4031, Basel, Switzerland.
11
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
12
Department of Neurology, University of Washington, Seattle, WA, 98195, USA.
13
Uppsala University, Department of Public Health and Caring Sciences, Geriatrics, Uppsala, Sweden.
14
Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA, USA.
15
Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, 02129, USA. cmaguire@mgh.harvard.edu.
16
Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA. cmaguire@mgh.harvard.edu.
17
Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA. bence.gyoergy@iob.ch.
18
Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, 02129, USA. bence.gyoergy@iob.ch.
19
Institute of Molecular and Clinical Ophthalmology Basel, 4031, Basel, Switzerland. bence.gyoergy@iob.ch.

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.

PMID:
31570731
PMCID:
PMC6769011
DOI:
10.1038/s41467-019-12449-2
[Indexed for MEDLINE]
Free PMC Article

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