Plant Molecular Biology

, Volume 78, Issue 4–5, pp 407–416 | Cite as

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

  • Lixin Li
  • Marek J. Piatek
  • Ahmed Atef
  • Agnieszka Piatek
  • Anjar Wibowo
  • Xiaoyun Fang
  • J. S. M. Sabir
  • Jian-Kang Zhu
  • Magdy M. Mahfouz
Article

Abstract

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

Keywords

Genome engineering TALE-based activators and repressors TALE nucleases (TALENs) Targeted mutagenesis Targeted activation and repression Genome modifications 

Supplementary material

11103_2012_9875_MOESM1_ESM.doc (176 kb)
Supplementary material 1 (DOC 176 kb)
11103_2012_9875_MOESM2_ESM.pdf (70 kb)
Supplementary material 2 (PDF 69 kb)
11103_2012_9875_MOESM3_ESM.pdf (960 kb)
Supplementary material 3 (PDF 960 kb)

References

  1. Boch J, Bonas U (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol 48:419–436PubMedCrossRefGoogle Scholar
  2. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512PubMedCrossRefGoogle Scholar
  3. Bogdanove AJ, Schornack S, Lahaye T (2010) TAL effectors: finding plant genes for disease and defense. Curr Opin Plant Biol 13:394–401PubMedCrossRefGoogle Scholar
  4. Bonas U, Stall RE, Staskawicz B (1989) Genetic and structural characterization of the avirulence gene AvrBs3 from Xanthomonas campestris pv. vesicatoria. Mol Gen Genet 218:127–136PubMedCrossRefGoogle Scholar
  5. Bonas U, Conrads-Strauch J, Balbo I (1993) Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3. Mol Gen Genet 238:261–269PubMedGoogle Scholar
  6. Cai CQ, Doyon Y, Ainley WM, Miller JC, Dekelver RC, Moehle EA, Rock JM, Lee YL, Garrison R, Schulenberg L et al (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol 69:699–709PubMedCrossRefGoogle Scholar
  7. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82PubMedCrossRefGoogle Scholar
  8. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761PubMedCrossRefGoogle Scholar
  9. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ et al (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26:702–708PubMedCrossRefGoogle Scholar
  10. Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S (2005) Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res 33:5978–5990PubMedCrossRefGoogle Scholar
  11. Fu F, Sander JD, Maeder M, Thibodeau-Beganny S, Joung JK, Dobbs D, Miller L, Voytas DF (2009) Zinc finger database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays. Nucleic Acids Res 37:D279–D283PubMedCrossRefGoogle Scholar
  12. Geissler R, Scholze H, Hahn S, Streubel J, Bonas U, Behrens SE, Boch J (2011) Transcriptional activators of human genes with programmable DNA-specificity. PLoS One 6:e19509PubMedCrossRefGoogle Scholar
  13. Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29:699–700PubMedCrossRefGoogle Scholar
  14. Kandavelou K, Ramalingam S, London V, Mani M, Wu J, Alexeev V, Civin CI, Chandrasegaran S (2009) Targeted manipulation of mammalian genomes using designed zinc finger nucleases. Biochem Biophys Res Commun 388:56–61PubMedCrossRefGoogle Scholar
  15. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B (2011a) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39:359–372PubMedCrossRefGoogle Scholar
  16. Li T, Huang S, Zhao X, Wright DA, Carpenter S, Spalding MH, Weeks DP, Yang B (2011b) Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 39:6315–6325PubMedCrossRefGoogle Scholar
  17. Mahfouz MM (2010) RNA-directed DNA methylation: mechanisms and functions. Plant Signal Behav 5:806–816PubMedCrossRefGoogle Scholar
  18. Mahfouz MM, Li L (2011) TALE nucleases and next generation GM crops. GM Crop 2:1236–1256Google Scholar
  19. Mahfouz MM, Li L, Piatek M, Fang X, Mansour H, Bangarusamy D, Zhu J-K (2011a) Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein. Plant Mol Biol. doi: 10.1007/s11103-011-9866-x
  20. Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A, Fang X, Zhu JK (2011b) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108:2623–2628PubMedCrossRefGoogle Scholar
  21. Mani M, Smith J, Kandavelou K, Berg JM, Chandrasegaran S (2005) Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem Biophys Res Commun 334:1191–1197PubMedCrossRefGoogle Scholar
  22. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785PubMedCrossRefGoogle Scholar
  23. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148PubMedCrossRefGoogle Scholar
  24. Morbitzer R, Romer P, Boch J, Lahaye T (2010) Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc Natl Acad Sci USA 107:21617–21622PubMedCrossRefGoogle Scholar
  25. Morbitzer R, Elsaesser J, Hausner J, Lahaye T (2011) Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res 39:5790–5799PubMedCrossRefGoogle Scholar
  26. Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326:1501PubMedCrossRefGoogle Scholar
  27. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 39(21):9283–9293Google Scholar
  28. Porteus M (2008) Design and testing of zinc finger nucleases for use in mammalian cells. Methods Mol Biol 435:47–61PubMedCrossRefGoogle Scholar
  29. Pruett-Miller SM, Reading DW, Porter SN, Porteus MH (2009) Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels. PLoS Genet 5:e1000376PubMedCrossRefGoogle Scholar
  30. Ramirez CL, Foley JE, Wright DA, Muller-Lerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA et al (2008) Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5:374–375PubMedCrossRefGoogle Scholar
  31. Romer P, Recht S, Lahaye T (2009) A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens. Proc Natl Acad Sci USA 106:20526–20531PubMedCrossRefGoogle Scholar
  32. Romer P, Recht S, Strauss T, Elsaesser J, Schornack S, Boch J, Wang S, Lahaye T (2010) Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae. New Phytol 187:1048–1057PubMedCrossRefGoogle Scholar
  33. Sander JD, Zaback P, Joung JK, Voytas DF, Dobbs D (2007) Zinc finger targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res 35:W599–W605PubMedCrossRefGoogle Scholar
  34. Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D (2010) ZiFiT (zinc finger targeter): an updated zinc finger engineering tool. Nucleic Acids Res 38:W462–W468PubMedCrossRefGoogle Scholar
  35. Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29:697–698PubMedCrossRefGoogle Scholar
  36. Scholze H, Boch J (2011) TAL effectors are remote controls for gene activation. Curr Opin Microbiol 14:47–53PubMedCrossRefGoogle Scholar
  37. Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L et al (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29:695–696PubMedCrossRefGoogle Scholar
  38. Weber E, Gruetzner R, Werner S, Engler C, Marillonnet S (2011) Assembly of designer TAL effectors by golden gate cloning. PLoS One 6:e19722PubMedCrossRefGoogle Scholar
  39. Weinthal D, Tovkach A, Zeevi V, Tzfira T (2010) Genome editing in plant cells by zinc finger nucleases. Trends Plant Sci 15(6):308–321PubMedCrossRefGoogle Scholar
  40. Wood AJ, Lo TW, Zeitler B, Pickle CS, Ralston EJ, Lee AH, Amora R, Miller JC, Leung E, Meng X et al (2011) Targeted genome editing across species using ZFNs and TALENs. Science 333:307PubMedCrossRefGoogle Scholar
  41. Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D, Christian M, Li X, Pierick CJ, Dobbs D, Peterson T et al (2010) High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci USA 107:12028–12033PubMedCrossRefGoogle Scholar
  42. Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29:149–153PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Lixin Li
    • 1
  • Marek J. Piatek
    • 1
  • Ahmed Atef
    • 2
  • Agnieszka Piatek
    • 1
  • Anjar Wibowo
    • 1
  • Xiaoyun Fang
    • 1
  • J. S. M. Sabir
    • 2
  • Jian-Kang Zhu
    • 3
  • Magdy M. Mahfouz
    • 1
  1. 1.Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and TechnologyThuwalKingdom of Saudi Arabia
  2. 2.Department of Biological Sciences, Faculty of SciencesKing Abdulaziz UniversityJeddahKingdom of Saudi Arabia
  3. 3.Department of Horticulture and Landscape ArchitecturePurdue UniversityWest LafayetteUSA

Personalised recommendations