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Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

Nature Biotechnology volume 33, pages 543548 (2015) | Download Citation

  • An Erratum to this article was published on 06 February 2018

This article has been updated

Abstract

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4–5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.

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Change history

  • 14 December 2017

    In the version of this article initially published, in the HTML only, in Online Methods “Cell culture and reagents,” in the last two sentences, “…supplied with 1 mM SCR7 inhibitor until analysis. At SCR7 concentrations of 60 mM and 10 mM….”, “mM,” in every case, should have been “μM.” The errors have been corrected in the HTML version of the article.

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Acknowledgements

We thank K. Petsch and H.P. Rahn from the FACS core facility for excellent technical support. This work was supported by the European Research Council (ERC advanced grant ERC-AG-LS6, to K.R.), the German Ministry of Education and Research within the VIP program (TAL-CUT 03V0261, to W.W. and R.K.) and the European Union within the EUCOMMTOOLS project (FP7-HEALTH-F4-2010-261492, to W.W.). The plasmids MSCV-IRES-mCherry and MSCV-IRES-BFP were kind gifts of F. Rosenbauer and M. Janz (Charite, Berlin). We are grateful to H. Ploegh for communicating to us unpublished results and support in coordinating publication.

Author information

Affiliations

  1. Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

    • Van Trung Chu
    • , Timm Weber
    • , Sandrine Sander
    • , Klaus Rajewsky
    •  & Ralf Kühn
  2. Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.

    • Benedikt Wefers
    • , Wolfgang Wurst
    •  & Ralf Kühn
  3. Deutsches Zentrum für Neurodegenerative Erkrankungen e.V., Munich, Germany.

    • Benedikt Wefers
    •  & Wolfgang Wurst
  4. Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universität München, Munich, Germany.

    • Wolfgang Wurst
  5. Berlin Institute of Health, Berlin, Germany.

    • Ralf Kühn

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Contributions

V.T.C., K.R. and R.K. conceived and designed the project, V.T.C. and T.W. acquired the data, V.T.C., K.R., T.W. and R.K. analyzed and interpreted the data, B.W., S.S. and W.W. provided materials, and V.T.C., K.R. and R.K. wrote the paper.

Competing interests

R.K. and W.W. are inventors on a patent application entitled "Improved recombination efficiency by inhibition of NHEJ DNA repair" (EP2718446).

Corresponding authors

Correspondence to Klaus Rajewsky or Ralf Kühn.

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DOI

https://doi.org/10.1038/nbt.3198

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