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Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription

Abstract

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.

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Figure 1: Design and construction of customized artificial TALEs for use in mammalian cells.
Figure 2: Functional characterization of the robustness of TALE-DNA recognition in mammalian cells and truncation analysis of TALE N- and C-termini.
Figure 3: Activation of endogenous genes in human cells by TALEs.

References

  1. Carr, P.A. & Church, G.M. Genome engineering. Nat. Biotechnol. 27, 1151–1162 (2009).

    CAS  Article  Google Scholar 

  2. Meister, G.E., Chandrasegaran, S. & Ostermeier, M. Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells. Nucleic Acids Res. 38, 1749–1759 (2010).

    CAS  Article  Google Scholar 

  3. Gonzalez, B. et al. Modular system for the construction of zinc-finger libraries and proteins. Nat. Protoc. 5, 791–810 (2010).

    CAS  Article  Google Scholar 

  4. Maeder, M.L., Thibodeau-Beganny, S., Sander, J.D., Voytas, D.F. & Joung, J.K. Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays. Nat. Protoc. 4, 1471–1501 (2009).

    CAS  Article  Google Scholar 

  5. Blancafort, P., Magnenat, L. & Barbas, C.F., III . Scanning the human genome with combinatorial transcription factor libraries. Nat. Biotechnol. 21, 269–274 (2003).

    CAS  Article  Google Scholar 

  6. Rosen, L.E. et al. Homing endonuclease I-CreI derivatives with novel DNA target specificities. Nucleic Acids Res. 34, 4791–4800 (2006).

    CAS  Article  Google Scholar 

  7. Grizot, S. et al. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. 37, 5405–5419 (2009).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  11. Bogdanove, A.J., Schornack, S. & Lahaye, T. TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol. 13, 394–401 (2010).

    CAS  Article  Google Scholar 

  12. Morbitzer, R., Romer, P., Boch, J. & Lahaye, T. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc. Natl. Acad. Sci. USA 107, 21617–21622 (2010).

    CAS  Article  Google Scholar 

  13. Kay, S., Hahn, S., Marois, E., Wieduwild, R. & Bonas, U. Detailed analysis of the DNA recognition motifs of the Xanthomonas type III effectors AvrBs3 and AvrBs3Deltarep16. Plant J. 59, 859–871 (2009).

    CAS  Article  Google Scholar 

  14. Romer, P. et al. Recognition of AvrBs3-like proteins is mediated by specific binding to promoters of matching pepper Bs3 alleles. Plant Physiol. 150, 1697–1712 (2009).

    Article  Google Scholar 

  15. Kay, S., Hahn, S., Marois, E., Hause, G. & Bonas, U. A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318, 648–651 (2007).

    CAS  Article  Google Scholar 

  16. Romer, P. et al. Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 318, 645–648 (2007).

    Article  Google Scholar 

  17. Scholze, H. & Boch, J. TAL effector-DNA specificity. Virulence 1, 428–432 (2010).

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  19. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. published online, doi:10.1038/nbt.1755 (22 December 2010).

  20. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4, e5553 (2009).

    Article  Google Scholar 

  21. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).

    Article  Google Scholar 

  22. Beerli, R.R., Segal, D.J., Dreier, B. & Barbas, C.F., III . Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl. Acad. Sci. USA 95, 14628–14633 (1998).

    CAS  Article  Google Scholar 

  23. Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J.A. & Kosik, K.S. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137, 647–658 (2009).

    CAS  Article  Google Scholar 

  24. Wei, Z. et al. Klf4 interacts directly with Oct4 and Sox2 to promote reprogramming. Stem Cells 27, 2969–2978 (2009).

    CAS  Article  Google Scholar 

  25. 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  Google Scholar 

  26. Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2010).

    Article  Google Scholar 

  27. Gordley, R.M., Gersbach, C.A. & Barbas, C.F., III . Synthesis of programmable integrases. Proc. Natl. Acad. Sci. USA 106, 5053–5058 (2009).

    CAS  Article  Google Scholar 

  28. Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the Harvard Society of Fellows (F.Z.), National Human Genome Research Institute Center for Excellence in Genomics Science (P50 HG003170, G.M.C.), Department of Energy Genomes to Life (DE-FG02-02ER63445, G.M.C.), Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.), the Wyss Institute for Biologically Inspired Engineering (G.M.C.) and National Institutes of Health Transformative R01 (R01 NS073124-01, F.Z. and P.A.). S.L. was partially supported by a predoctoral fellowship from the European School of Molecular Medicine (S.E.M.M.). We thank the entire Church and Arlotta laboratories for discussion and support.

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Authors

Contributions

F.Z. and L.C. conceived the study. F.Z., L.C., S.L. and S.K. designed, performed and analyzed all experiments. P.A. supervised the work of S.L. and G.M.C. supervised the work of F.Z., L.C. and S.K. G.M.C., P.A. and F.Z. provided support for this study. F.Z., L.C. and P.A. wrote the manuscript with support from all authors. G.M.C and P.A. equally contributed to this work.

Corresponding authors

Correspondence to Feng Zhang or Paola Arlotta.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Figs. 1–3, Supplementary Methods and Supplementary Sequences (PDF 804 kb)

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Zhang, F., Cong, L., Lodato, S. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29, 149–153 (2011). https://doi.org/10.1038/nbt.1775

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