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

Author

Listed:
  • Killian S. Hanlon

    (Harvard Medical School
    Massachusetts General Hospital)

  • Benjamin P. Kleinstiver

    (Massachusetts General Hospital
    Massachusetts General Hospital
    Harvard Medical School)

  • Sara P. Garcia

    (Harvard Medical School
    Massachusetts General Hospital
    Massachusetts General Hospital)

  • Mikołaj P. Zaborowski

    (Massachusetts General Hospital
    Harvard Medical School
    Poznań University of Medical Sciences)

  • Adrienn Volak

    (Massachusetts General Hospital
    Institute of Molecular and Clinical Ophthalmology Basel)

  • 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
    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
    Massachusetts General Hospital
    Massachusetts General Hospital
    Harvard T. H. Chan School of Public Health)

  • J. Keith Joung

    (Harvard Medical School
    Massachusetts General Hospital
    Massachusetts General Hospital)

  • Xandra O. Breakefield

    (Massachusetts General Hospital
    Harvard Medical School)

  • Casey A. Maguire

    (Massachusetts General Hospital
    Harvard Medical School)

  • Bence György

    (Harvard Medical School
    Massachusetts General Hospital
    Institute of Molecular and Clinical Ophthalmology Basel)

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.

Suggested Citation

  • 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 & , 2019. "High levels of AAV vector integration into CRISPR-induced DNA breaks," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-12449-2
    DOI: 10.1038/s41467-019-12449-2
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    Cited by:

    1. Xiangjun He & Zhenjie Zhang & Junyi Xue & Yaofeng Wang & Siqi Zhang & Junkang Wei & Chenzi Zhang & Jue Wang & Brian Anugerah Urip & Chun Christopher Ngan & Junjiang Sun & Yuefeng Li & Zhiqian Lu & Hui, 2022. "Low-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    2. Jingjing Ren & Xiaofeng Liao & Julia M. Lewis & Jungsoo Chang & Rihao Qu & Kacie R. Carlson & Francine Foss & Michael Girardi, 2024. "Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    3. Jianhang Yin & Kailun Fang & Yanxia Gao & Liqiong Ou & Shaopeng Yuan & Changchang Xin & Weiwei Wu & Wei-wei Wu & Jiaxu Hong & Hui Yang & Jiazhi Hu, 2022. "Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    4. Changchang Xin & Jianhang Yin & Shaopeng Yuan & Liqiong Ou & Mengzhu Liu & Weiwei Zhang & Jiazhi Hu, 2022. "Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    5. Milan Gautam & Antony Jozic & Grace Li-Na Su & Marco Herrera-Barrera & Allison Curtis & Sebastian Arrizabalaga & Wayne Tschetter & Renee C. Ryals & Gaurav Sahay, 2023. "Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    6. Lisa Maria Riedmayr & Klara Sonnie Hinrichsmeyer & Stefan Bernhard Thalhammer & David Manuel Mittas & Nina Karguth & Dina Yehia Otify & Sybille Böhm & Valentin Johannes Weber & Michael David Bartosche, 2023. "mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    7. Eriya Kenjo & Hiroyuki Hozumi & Yukimasa Makita & Kumiko A. Iwabuchi & Naoko Fujimoto & Satoru Matsumoto & Maya Kimura & Yuichiro Amano & Masataka Ifuku & Youichi Naoe & Naoto Inukai & Akitsu Hotta, 2021. "Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
    8. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    9. Xiang Meng & Ruixuan Jia & Xinping Zhao & Fan Zhang & Shaohong Chen & Shicheng Yu & Xiaozhen Liu & Hongliang Dou & Xuefeng Feng & Jinlu Zhang & Ni Wang & Boling Xu & Liping Yang, 2024. "In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    10. Jianli Tao & Qi Wang & Carlos Mendez-Dorantes & Kathleen H. Burns & Roberto Chiarle, 2022. "Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    11. Ron Baik & M. Kyle Cromer & Steve E. Glenn & Christopher A. Vakulskas & Kay O. Chmielewski & Amanda M. Dudek & William N. Feist & Julia Klermund & Suzette Shipp & Toni Cathomen & Daniel P. Dever & Mat, 2024. "Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    12. Raed Ibraheim & Phillip W. L. Tai & Aamir Mir & Nida Javeed & Jiaming Wang & Tomás C. Rodríguez & Suk Namkung & Samantha Nelson & Eraj Shafiq Khokhar & Esther Mintzer & Stacy Maitland & Zexiang Chen &, 2021. "Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo," Nature Communications, Nature, vol. 12(1), pages 1-17, December.

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