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

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High levels of AAV vector integration into CRISPR-induced DNA breaks.
Nature Communications ( IF 14.7 ) Pub Date : 2019-09-30 , DOI: 10.1038/s41467-019-12449-2
Killian S Hanlon _{1,

2} , Benjamin P Kleinstiver _{3,

4,

5} , Sara P Garcia _{5,

6,

7} , Mikołaj P Zaborowski _{2,

8,

9} , Adrienn Volak _{2,

10} , Stefan E Spirig ₁₀ , Alissa Muller ₁₀ , Alexander A Sousa _{6,

7} , Shengdar Q Tsai ₁₁ , Niclas E Bengtsson ₁₂ , Camilla Lööv ₁₃ , Martin Ingelsson ₁₃ , Jeffrey S Chamberlain ₁₂ , David P Corey ₁ , Martin J Aryee _{5,

6,

7,

14} , J Keith Joung _{5,

6,

7} , Xandra O Breakefield _{2,

8} , Casey A Maguire _{2,

8} , Bence György _{1,

2,

10}

Affiliation

Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA.
Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, 02129, USA.
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
Department of Pathology, Harvard Medical School, Boston, MA, USA.
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, 02129, USA.
Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA.
Department of Gynecology, Obstetrics and Gynecologic Oncology, Division of Gynecologic Oncology, Poznań University of Medical Sciences, 60-535, Poznań, Poland.
Institute of Molecular and Clinical Ophthalmology Basel, 4031, Basel, Switzerland.
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Department of Neurology, University of Washington, Seattle, WA, 98195, USA.
Uppsala University, Department of Public Health and Caring Sciences, Geriatrics, Uppsala, Sweden.
Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA, USA.

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.

中文翻译：

AAV 载体高水平整合到 CRISPR 诱导的 DNA 断裂中。

腺相关病毒 (AAV) 载体在临床前模型中显示出有希望的结果，但编码 CRISPR-Cas 核酸酶的 AAV 载体转导的基因组后果仍在研究中。在这项研究中，我们观察到培养的小鼠神经元、小鼠大脑、肌肉和耳蜗中治疗相关基因中 AAV 高水平整合（高达 47%）到 Cas9 诱导的双链断裂 (DSB) 中。小鼠大脑中的全基因组 AAV 作图显示，除 CRISPR/Cas9 靶位点外，AAV 整合没有总体增加。为了详细表征整合事件，我们设计了编码 465 bp λ 噬菌体 DNA (AAV-λ465) 的微型 AAV，从而能够对整个整合载体基因组进行测序。培养细胞中 AAV-465λ 的整合概况显示 Cas9 靶位点的全长和片段 AAV 基因组。我们的数据表明，AAV 整合应被视为利用 AAV 进行基因组编辑的应用程序的常见结果。

更新日期：2019-09-30

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