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Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

Geoffrey L. Rogers, Hsu-Yu Chen, Heidy Morales, View ORCID ProfilePaula M. Cannon
doi: https://doi.org/10.1101/704197
Geoffrey L. Rogers
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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Hsu-Yu Chen
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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Heidy Morales
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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Paula M. Cannon
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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  • ORCID record for Paula M. Cannon
  • For correspondence: pcannon@usc.edu
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Abstract

Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 21, 2019.
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Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break
Geoffrey L. Rogers, Hsu-Yu Chen, Heidy Morales, Paula M. Cannon
bioRxiv 704197; doi: https://doi.org/10.1101/704197
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Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break
Geoffrey L. Rogers, Hsu-Yu Chen, Heidy Morales, Paula M. Cannon
bioRxiv 704197; doi: https://doi.org/10.1101/704197

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