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.

  • Resource
  • Published:

A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes

Nature Biotechnology volume 31, pages 76–81 (2013)Cite this article

Subjects

Abstract

Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The LIC assembly approach to generating TALEN genes.
Figure 2: LIC assembly and validation of a TALEN targeting STAT6.
Figure 3: Large-scale assembly of TALENs using LIC.
Figure 4: Evaluation of targeting activity dependence on spacer length and RVD composition.
Figure 5: A 5-mer library for LIC assembly of TALENs.
Figure 6: On target activity of 15.5-RVD LIC TALENs.

Similar content being viewed by others

References

  1. Boch, J. & Bonas, U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 48, 419–436 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Carroll, D. Genome engineering with zinc-finger nucleases. Genetics 188, 773–782 (2011).

    Article  CAS  Google Scholar 

  6. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. Liu, J. et al. Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J. Genet. Genomics 39, 209–215 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).

    Article  CAS  Google Scholar 

  13. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

    Article  CAS  Google Scholar 

  14. Watanabe, T. et al. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat. Commun. 3, 1017 (2012).

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Geissler, R. et al. Transcriptional activators of human genes with programmable DNA-specificity. PLoS ONE 6, e19509 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Aslanidis, C. & de Jong, P.J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074 (1990).

    Article  CAS  Google Scholar 

  22. Geu-Flores, F., Nour-Eldin, H.H., Nielsen, M.T. & Halkier, B.A. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 35, e55 (2007).

    Article  Google Scholar 

  23. Schmid-Burgk, J.L. et al. Rapid hierarchical assembly of medium-size DNA cassettes. Nucleic Acids Res. 40, e92 (2012).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  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).

    Article  CAS  Google Scholar 

  26. Streubel, J., Blucher, C., Landgraf, A. & Boch, J. TAL effector RVD specificities and efficiencies. Nat. Biotechnol. 30, 593–595 (2012).

    Article  CAS  Google Scholar 

  27. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Cathomen for technical advice with the T7EI assay and M. Hölzel for helpful discussion. V.H. is member of the excellence cluster ImmunoSensation and supported by grants from the German Research Foundation (SFB704 and SFB670) and the European Research Council (ERC-2009-StG 243046).

Author information

Author notes

  1. Jonathan L Schmid-Burgk and Tobias Schmidt: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Clinical Chemistry and Clinical Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Bonn, Germany

    Jonathan L Schmid-Burgk, Tobias Schmidt, Vera Kaiser, Klara Höning & Veit Hornung

Authors
  1. Jonathan L Schmid-Burgk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tobias Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vera Kaiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Klara Höning
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Veit Hornung
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.L.S.-B., T.S. and V.H. developed the methodology, designed experiments, analyzed the data and wrote the manuscript. J.L.S.-B., T.S., V.K. and K.H. performed the experiments. V.H. supervised the project.

Corresponding author

Correspondence to Veit Hornung.

Ethics declarations

Competing interests

J.L.S.-B., T.S. and V.H are inventors on a patent application dealing with LIC assembly of TALE genes.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–5, Supplementary Methods and Supplementary Note 1 (PDF 4128 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmid-Burgk, J., Schmidt, T., Kaiser, V. et al. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes. Nat Biotechnol 31, 76–81 (2013). https://doi.org/10.1038/nbt.2460

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2460

This article is cited by

Search

Advanced 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