Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA

Alex N. Zelensky, Joost Schimmel, Hanneke Kool, Roland Kanaar () and Marcel Tijsterman ()
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Alex N. Zelensky: Erasmus University Medical Centre
Joost Schimmel: Leiden University Medical Centre
Hanneke Kool: Leiden University Medical Centre
Roland Kanaar: Erasmus University Medical Centre
Marcel Tijsterman: Leiden University Medical Centre

Nature Communications, 2017, vol. 8, issue 1, 1-7

Abstract: Abstract Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.

Date: 2017
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DOI: 10.1038/s41467-017-00124-3

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