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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Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).
Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).
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).
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).
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).
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).
Wu, Y. et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).
Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Panier, S. & Boulton, S.J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).
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).
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).
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).
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).
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).
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).
Srivastava, M. et al. An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Cell 151, 1474–1487 (2012).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Song, L., Florea, L. & Langmead, B. Lighter: fast and memory-efficient sequencing error correction without counting. Genome Biol. 15, 509 (2014).
Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).
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).
Hailemariam, T.K. et al. Sphingomyelin synthase 2 deficiency attenuates NFkappaB activation. Arterioscler. Thromb. Vasc. Biol. 28, 1519–1526 (2008).
Singh, P., Schimenti, J.C. & Bolcun-Filas, E. A mouse geneticists's practical guide to CRISPR applications. Genetics 199, 1–15 (2015).
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.).
The authors declare no competing financial interests.
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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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