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Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining

Nature Biotechnology volume 33, pages 538–542 (2015)Cite this article

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A Corrigendum to this article was published on 05 February 2016

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Abstract

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.

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Figure 1: The NHEJ inhibitor Scr7 enhances the efficiency of insertional mutagenesis in cell lines.
Figure 2: Co-injection of Scr7 enhances the efficiency of precise genome editing in mouse embryos.

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Change history

  • 20 April 2015

    In the version of this article initially published online, the received date of the paper was given as 17 October 2014; the correct date is 11 July 2014. On p.4, left column, second paragraph, 1 μM Scr7 should have been 1 mM Scr7; in Online Methods, second paragraph, Cas9 mRNA, sgRNA and template oligos were erroneously given in ng/ml rather than ng/μl. A technique was misidentified as ChIP; in all cases it should be “chip.” The possibility of using Scr7 as a means of enhancing homology-directed repair was recently also mentioned in a review article that was published while our manuscript was under review. A reference to this article has been added to the manuscript. Finally, the P values in Table 1 for Kell and Lgkc were reversed and should be *P<0.05 (Igkc), ***P<0.005 (Kell) and not *P<0.05 (Kell), ***P<0.005 (Igkc). The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Ploegh laboratory, especially N. Pishesha and F. Tafesse, for critical reading of the manuscript; P.A. Koenig, L.K. Swee, C.S. Shivalila, H. Wang, H. Yang and R. Jaenisch for discussions; T. Wang and D.M. Sabatini for pCW-Cas9; P. Thiru and G. Bell of BARC (WIBR) for assistance with statistical analysis; and F. Zhang (Addgene) for pX330. This work was supported by the National Institutes of Health (RO1 grant AI087879-01 to H.L.P.), Japan Society for the Promotion of Science (to T.M.), Japan Heart Foundation (to T.M.), AACR-Pancreatic Cancer Action Network (to S.K.D. and H.L.P.) and an SNSF Early Postdoc Mobility fellowship (to M.C.T.).

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Takeshi Maruyama, Stephanie K Dougan, Matthias C Truttmann, Angelina M Bilate, Jessica R Ingram & Hidde L Ploegh

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Hidde L Ploegh

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  1. Takeshi Maruyama
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Contributions

T.M. and H.L.P. conceived of and designed experiments; T.M., S.K.D. and A.M.B. performed experiments; T.M. and M.T. analyzed data; T.M., J.R.I. and H.L.P. wrote the manuscript.

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Correspondence to Hidde L Ploegh.

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Supplementary Figures 1–9, Supplementary Tables 1–7 (PDF 16545 kb)

Supplementary Protocol

CellProfiler pipeline protocol (TXT 9 kb)

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Maruyama, T., Dougan, S., Truttmann, M. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 33, 538–542 (2015). https://doi.org/10.1038/nbt.3190

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