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

Killian S. Hanlon, Benjamin P. Kleinstiver, Sara P. Garcia, Mikołaj P. Zaborowski, Adrienn Volak, Stefan E. Spirig, Alissa Muller, Alexander A. Sousa, Shengdar Q. Tsai, Niclas E. Bengtsson, Camilla Lööv, Martin Ingelsson, Jeffrey S. Chamberlain, David P. Corey, Martin J. Aryee, J. Keith Joung, Xandra O. Breakefield, Casey A. Maguire () and Bence György ()
Additional contact information
Killian S. Hanlon: Harvard Medical School
Benjamin P. Kleinstiver: Massachusetts General Hospital
Sara P. Garcia: Harvard Medical School
Mikołaj P. Zaborowski: Massachusetts General Hospital
Adrienn Volak: Massachusetts General Hospital
Stefan E. Spirig: Institute of Molecular and Clinical Ophthalmology Basel
Alissa Muller: Institute of Molecular and Clinical Ophthalmology Basel
Alexander A. Sousa: Massachusetts General Hospital
Shengdar Q. Tsai: St. Jude Children’s Research Hospital
Niclas E. Bengtsson: University of Washington
Camilla Lööv: Uppsala University, Department of Public Health and Caring Sciences, Geriatrics
Martin Ingelsson: Uppsala University, Department of Public Health and Caring Sciences, Geriatrics
Jeffrey S. Chamberlain: University of Washington
David P. Corey: Harvard Medical School
Martin J. Aryee: Harvard Medical School
J. Keith Joung: Harvard Medical School
Xandra O. Breakefield: Massachusetts General Hospital
Casey A. Maguire: Massachusetts General Hospital
Bence György: Harvard Medical School

Nature Communications, 2019, vol. 10, issue 1, 1-11

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-12449-2

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