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.

A library of TAL effector nucleases spanning the human genome

Abstract

Transcription activator–like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Optimization of TALEN architecture.
Figure 2: Assembly of TALEN plasmids using a one-step Golden-Gate cloning system.
Figure 3: Targeted gene-disrupting activities of TALENs.
Figure 4: TALEN-mediated chromosomal deletions.
Figure 5: NF-κB-associated gene-knockout cell lines.

References

  1. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    CAS  Article  PubMed  Google Scholar 

  2. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    CAS  Article  PubMed  Google Scholar 

  3. Krueger, U. et al. Insights into effective RNAi gained from large-scale siRNA validation screening. Oligonucleotides 17, 237–250 (2007).

    CAS  Article  PubMed  Google Scholar 

  4. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    CAS  Article  PubMed  Google Scholar 

  5. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    CAS  Article  PubMed  Google Scholar 

  6. Khan, A.A. et al. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat. Biotechnol. 27, 549–555 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H. & Williams, B.R. Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839 (2003).

    CAS  Article  PubMed  Google Scholar 

  8. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317, 230–234 (1985).

    CAS  Article  PubMed  Google Scholar 

  9. Bylund, L., Kytola, S., Lui, W.O., Larsson, C. & Weber, G. Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches. Cytogenet. Genome Res. 106, 28–32 (2004).

    CAS  Article  PubMed  Google Scholar 

  10. Macville, M. et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res. 59, 141–150 (1999).

    CAS  PubMed  Google Scholar 

  11. Mali, P. et al. RNA-Guided Human Genome Engineering via Cas9. Science doi:10.1126/science.1232033 (3 January 2013).

  12. Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    CAS  Article  PubMed  Google Scholar 

  13. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

    CAS  Article  PubMed  Google Scholar 

  14. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    CAS  Article  PubMed  Google Scholar 

  15. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science doi:10.1126/science.1231143 (3 January 2013).

  16. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. advance online publication, doi:10.1038/nbt.2501 (29 January 2013).

  17. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. advance online publication, doi:10.1038/nbt.2507 (29 January 2013).

  18. Kim, H.J., Lee, H.J., Kim, H., Cho, S.W. & Kim, J.S. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 19, 1279–1288 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Kim, Y.G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Kim, J.S., Lee, H.J. & Carroll, D. Genome editing with modularly assembled zinc-finger nucleases. Nat. Methods 7, 91 author reply 91–92 (2010).

    CAS  Article  PubMed  Google Scholar 

  22. Gupta, A. et al. An optimized two-finger archive for ZFN-mediated gene targeting. Nat. Methods 9, 588–590 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Carlson, D.F. et al. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl. Acad. Sci. USA 109, 17382–17387 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Cade, L. et al. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic Acids Res. 40, 8001–8010 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Kim, H., Um, E., Cho, S.R., Jung, C. & Kim, J.S. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat. Methods 8, 941–943 (2011).

    CAS  Article  PubMed  Google Scholar 

  27. Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res. 39, 6315–6325 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Morbitzer, R., Elsaesser, J., Hausner, J. & Lahaye, T. Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res. 39, 5790–5799 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Weber, E., Gruetzner, R., Werner, S., Engler, C. & Marillonnet, S. Assembly of designer TAL effectors by Golden Gate cloning. PLoS ONE 6, e19722 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Guo, J., Gaj, T. & Barbas, C.F. 3rd Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J. Mol. Biol. 400, 96–107 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    CAS  Article  PubMed  Google Scholar 

  34. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    CAS  Article  PubMed  Google Scholar 

  35. Seal, R.L., Gordon, S.M., Lush, M.J., Wright, M.W. & Bruford, E.A. genenames.org: the HGNC resources in 2011. Nucleic Acids Res. 39, D514–D519 (2011).

    CAS  Article  PubMed  Google Scholar 

  36. Pruitt, K.D., Tatusova, T., Brown, G.R. & Maglott, D.R. NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy. Nucleic Acids Res. 40, D130–D135 (2012).

    CAS  Article  PubMed  Google Scholar 

  37. Valton, J. et al. Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation. J. Biol. Chem. 287, 38427–38432 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Deng, D. et al. Recognition of methylated DNA by TAL effectors. Cell Res. 22, 1502–1504 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Bultmann, S. et al. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers. Nucleic Acids Res. 40, 5368–5377 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–9293 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Pattanayak, V., Ramirez, C.L., Joung, J.K. & Liu, D.R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    CAS  PubMed  Google Scholar 

  43. Lee, H.J., Kim, E. & Kim, J.S. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 20, 81–89 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 (2011).

    CAS  Article  PubMed  Google Scholar 

  45. Huang, P. et al. Heritable gene targeting in zebrafish using customized TALENs. Nat. Biotechnol. 29, 699–700 (2011).

    Article  PubMed  Google Scholar 

  46. Sander, J.D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697–698 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Li, T., Liu, B., Spalding, M.H., Weeks, D.P. & Yang, B. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 30, 390–392 (2012).

    CAS  Article  PubMed  Google Scholar 

  48. Cornu, T.I. et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358 (2008).

    CAS  Article  PubMed  Google Scholar 

  49. Perkins, N.D. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat. Rev. Mol. Cell Biol. 8, 49–62 (2007).

    CAS  Article  PubMed  Google Scholar 

  50. Volcic, M. et al. NF-kappaB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes. Nucleic Acids Res. 40, 181–195 (2012).

    CAS  Article  PubMed  Google Scholar 

  51. Gewurz, B.E. et al. Genome-wide siRNA screen for mediators of NF-kappaB activation. Proc. Natl. Acad. Sci. USA 109, 2467–2472 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Briggs, A.W. et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res. 40, e117 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Kim, S., Lee, M.J., Kim, H., Kang, M. & Kim, J.S. Preassembled zinc-finger arrays for rapid construction of ZFNs. Nat. Methods 8, 7 (2011).

    CAS  Article  PubMed  Google Scholar 

  54. Sigoillot, F.D. & King, R.W. Vigilance and validation: Keys to success in RNAi screening. ACS Chem. Biol. 6, 47–60 (2011).

    CAS  Article  PubMed  Google Scholar 

  55. Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Adamson, B., Smogorzewska, A., Sigoillot, F.D., King, R.W. & Elledge, S.J. A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat. Cell Biol. 14, 318–328 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Holt, N. et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 28, 839–847 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Research Foundation of Korea (J.-S.K., 2012-0001225), the Intelligent Synthetic Biology Center of the Global Frontier Project funded by the Ministry of Education, Science and Technology, Korea (D.B., 2011-0031956), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (S.K., 311062-04-2-sb1010), and Plant Molecular Breeding Center of Next-Generation BioGreen 21 Program (S.K., PJ009081).

Author information

Authors and Affiliations

Authors

Contributions

J.-S.K., S.K. and D.B. supervised the research and wrote the manuscript. All the other authors performed the experiments.

Corresponding authors

Correspondence to Seokjoong Kim or Jin-Soo Kim.

Ethics declarations

Competing interests

J.Y.Y., M.S.L., E.M.G., H.J.S. and S.K. are employees of ToolGen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 2–6 and Supplementary Methods (PDF 2293 kb)

Supplementary Table 1

Supplementary Table 1 (XLSX 100 kb)

Supplementary Table 7

Supplementary Table 7 (XLSX 1501 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kim, Y., Kweon, J., Kim, A. et al. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31, 251–258 (2013). https://doi.org/10.1038/nbt.2517

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2517

This article is cited by

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