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A TALE nuclease architecture for efficient genome editing

Nature Biotechnology volume 29, pages 143–148 (2011)Cite this article

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Abstract

Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator–like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

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Figure 1: Structure and DNA-binding specificity of TALE proteins.
Figure 2: Activation of the endogenous human NTF3 gene by engineered TALE transcription factors.
Figure 3: Modification of the endogenous NTF3 locus in human cells.
Figure 4: Modification of endogenous human CCR5.
Figure 5: TALEN-mediated gene editing by HDR.
Figure 6: Base preferences of the five RVDs used in this study, as determined by averaging SELEX-derived base preferences for NT-L (Fig. 2e) and NT-R (Supplementary Fig. 5), as well as two additional designed TALEs “VEGF-1” and “CCR5-1” (Supplementary Fig. 10).

References

  1. 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).

    Article  CAS  PubMed  Google Scholar 

  2. Davis, D. & Stokoe, D. Zinc finger nucleases as tools to understand and treat human diseases. BMC Med. 8, 42 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Camenisch, T.D., Brilliant, M.H. & Segal, D.J. Critical parameters for genome editing using zinc finger nucleases. Mini Rev. Med. Chem. 8, 669–676 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Carroll, D. Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther. 15, 1463–1468 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cathomen, T. & Joung, J.K. Zinc-finger nucleases: the next generation emerges. Mol. Ther. 16, 1200–1207 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Galetto, R., Duchateau, P. & Paques, F. Targeted approaches for gene therapy and the emergence of engineered meganucleases. Expert Opin. Biol. Ther. 9, 1289–1303 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Hoeijmakers, J.H. Genome maintenance mechanisms for preventing cancer. Nature 411, 366–374 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Ochiai, H. et al. Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15, 875–885 (2010).

    CAS  PubMed  Google Scholar 

  9. Takasu, Y. et al. Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem. Mol. Biol. 40, 759–765 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Morton, J., Davis, M.W., Jorgensen, E.M. & Carroll, D. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Proc. Natl. Acad. Sci. USA 103, 16370–16375 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Benabdallah, B.F. et al. Targeted gene addition to human mesenchymal stromal cells as a cell-based plasma-soluble protein delivery platform. Cytotherapy 12, 394–399 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Bibikova, M., Golic, M., Golic, K.G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  PubMed  Google Scholar 

  20. Maeder, M.L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  26. 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).

    Article  CAS  PubMed  Google Scholar 

  27. Kay, S. & Bonas, U. How Xanthomonas type III effectors manipulate the host plant. Curr. Opin. Microbiol. 12, 37–43 (2009).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. published online, doi:10.1093/nar/gkq704 (10 August 2010).

  32. Szurek, B., Rossier, O., Hause, G. & Bonas, U. Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell. Mol. Microbiol. 46, 13–23 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Chao, M.V., Rajagopal, R. & Lee, F.S. Neurotrophin signalling in health and disease. Clin. Sci. (Lond.) 110, 167–173 (2006).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  36. Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. Doyon, Y. et al. Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nat. Methods 7, 459–460 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Jeggo, P.A. DNA breakage and repair. Adv. Genet. 38, 185–218 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Orlando, S.J. et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 38, e152 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Liu, R. et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367–377 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Townsend, J.A. et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442–445 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Redondo, P. et al. Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456, 107–111 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. DeKelver, R.C. et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 20, 1133–1142 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Doyon, Y. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods published online, doi: 10.1038/nmeth.1539 (5 December 2010).

  45. Cuthbert, G.L. et al. Histone deimination antagonizes arginine methylation. Cell 118, 545–553 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Reik, A. et al. Enhanced protein production by engineered zinc finger proteins. Biotechnol. Bioeng. 97, 1180–1189 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Liu, P.Q. et al. Isogenic human cell lines for drug discovery: regulation of target gene expression by engineered zinc-finger protein transcription factors. J. Biomol. Screen. 10, 304–313 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Whitlock, P.R., Hackett, N.R., Leopold, P.L., Rosengart, T.K. & Crystal, R.G. Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs. Mol. Ther. 9, 67–75 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Ozawa, C.R. et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J. Clin. Invest. 113, 516–527 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Tan, S. et al. Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity. Proc. Natl. Acad. Sci. USA 100, 11997–12002 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Morbitzer, R., Römer, 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 (2010).

  52. Hoover, D.M. & Lubkowski, J. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res. 30, e43 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Abrahamson for critically reviewing this manuscript, and also E. Wolffe and D. Guschin for helpful comments and suggestions. We also thank S. Orlando, Y. Santiago, S. Lussier, A. Vincent and S. Lam for technical support.

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Author notes

  1. Jeffrey C Miller and Siyuan Tan: These authors contributed equally to this work.

Authors and Affiliations

  1. Sangamo BioSciences, Inc., Richmond, California, USA

    Jeffrey C Miller, Siyuan Tan, Guijuan Qiao, Kyle A Barlow, Jianbin Wang, Danny F Xia, Xiangdong Meng, David E Paschon, Elo Leung, Sarah J Hinkley, Gladys P Dulay, Kevin L Hua, Irina Ankoudinova, Gregory J Cost, Fyodor D Urnov, H Steve Zhang, Michael C Holmes, Lei Zhang, Philip D Gregory & Edward J Rebar

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Contributions

J.C.M. and S.T. designed studies, performed experiments and analyzed data. G.Q., K.A.B., J.W., D.F.X., X.M., D.E.P., K.L.H., and G.J.C. performed studies. S.T, G.Q., E.L., S.J.H., G.P.D., L.Z., and I.A. developed new procedures and assembled constructs. F.D.U., H.S.Z, M.C.H., L.Z., P.D.G. and E.J.R. supervised studies and designed experiments. J.C.M., P.D.G. and E.J.R. wrote the manuscript.

Corresponding author

Correspondence to Edward J Rebar.

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All authors are current or past full-time employees of Sangamo BioSciences, Inc.

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Supplementary Figs. 1–12 and Supplementary Methods (PDF 1093 kb)

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Miller, J., Tan, S., Qiao, G. et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29, 143–148 (2011). https://doi.org/10.1038/nbt.1755

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