Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

A Corrigendum to this article was published on 05 February 2016

This article has been updated

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

Primary accessions

Sequence Read Archive

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.

References

  1. 1

    Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  Article  Google Scholar 

  3. 3

    Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  Article  Google Scholar 

  4. 4

    Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    CAS  Article  Google Scholar 

  5. 5

    Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Wu, Y. et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).

    CAS  Article  Google Scholar 

  8. 8

    Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Panier, S. & Boulton, S.J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Pierce, A.J., Hu, P., Han, M., Ellis, N. & Jasin, M. Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev. 15, 3237–3242 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Boboila, C. et al. Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4. J. Exp. Med. 207, 417–427 (2010).

    Article  Google Scholar 

  13. 13

    Frit, P., Barboule, N., Yuan, Y., Gomez, D. & Calsou, P. Alternative end-joining pathway(s): bricolage at DNA breaks. DNA Repair (Amst.) 17, 81–97 (2014).

    CAS  Article  Google Scholar 

  14. 14

    Beumer, K.J., Trautman, J.K., Mukherjee, K. & Carroll, D. Donor DNA Utilization during gene targeting with zinc-finger nucleases. G3 3, 657–664 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Adachi, N., Ishino, T., Ishii, Y., Takeda, S. & Koyama, H. DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair. Proc. Natl. Acad. Sci. USA 98, 12109–12113 (2001).

    CAS  Article  Google Scholar 

  16. 16

    Frank, K.M. et al. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Srivastava, M. et al. An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Cell 151, 1474–1487 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Sanyal, S. et al. Type I interferon imposes a TSG101/ISG15 checkpoint at the Golgi for glycoprotein trafficking during influenza virus infection. Cell Host Microbe 14, 510–521 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Van Kaer, L., Ashton-Rickardt, P.G., Ploegh, H.L. & Tonegawa, S. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4–8+ T cells. Cell 71, 1205–1214 (1992).

    CAS  Article  Google Scholar 

  20. 20

    Christianson, J.C., Shaler, T.A., Tyler, R.E. & Kopito, R.R. OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1–SEL1L ubiquitin ligase complex for ERAD. Nat. Cell Biol. 10, 272–282 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Lee, C.C., Freinkman, E., Sabatini, D.M. & Ploegh, H.L. The protein synthesis inhibitor blasticidin S enters mammalian cells via leucine-rich repeat-containing protein 8D. J. Biol. Chem. 289, 17124–17131 (2014).

    CAS  Article  Google Scholar 

  22. 22

    Rinkevich, Y. et al. Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature. Nat. Cell Biol. 14, 1251–1260 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Hovenden, M. et al. IgG subclass and heavy chain domains contribute to binding and protection by mAbs to the poly gamma-D-glutamic acid capsular antigen of Bacillus anthracis. PLoS Pathog. 9, e1003306 (2013).

    CAS  Article  Google Scholar 

  24. 24

    Shi, J. et al. Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes. Proc. Natl. Acad. Sci. USA 111, 10131–10136 (2014).

    CAS  Article  Google Scholar 

  25. 25

    So, S., Adachi, N., Lieber, M.R. & Koyama, H. Genetic interactions between BLM and DNA ligase IV in human cells. J. Biol. Chem. 279, 55433–55442 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Simsek, D. & Jasin, M. Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation. Nat. Struct. Mol. Biol. 17, 410–416 (2010).

    CAS  Article  Google Scholar 

  27. 27

    Song, L., Florea, L. & Langmead, B. Lighter: fast and memory-efficient sequencing error correction without counting. Genome Biol. 15, 509 (2014).

    Article  Google Scholar 

  28. 28

    Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    CAS  Article  Google Scholar 

  30. 30

    Popp, M.W., Antos, J.M. & Ploegh, H.L. Site-specific protein labeling via sortase-mediated transpeptidation. Curr. Protoc. Protein Sci. Chapter 15 Unit 15 13 (2009).

  31. 31

    Hailemariam, T.K. et al. Sphingomyelin synthase 2 deficiency attenuates NFkappaB activation. Arterioscler. Thromb. Vasc. Biol. 28, 1519–1526 (2008).

    CAS  Article  Google Scholar 

  32. 32

    Singh, P., Schimenti, J.C. & Bolcun-Filas, E. A mouse geneticists's practical guide to CRISPR applications. Genetics 199, 1–15 (2015).

    CAS  Article  Google Scholar 

Download references

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.).

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Hidde L Ploegh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Supplementary Protocol

CellProfiler pipeline protocol (TXT 9 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing