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Current Genetics

, Volume 60, Issue 2, pp 61–74 | Cite as

Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae

  • Mustapha Aouida
  • Marek J. Piatek
  • Dhinoth K. Bangarusamy
  • Magdy M. MahfouzEmail author
Original Paper

Abstract

The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects.

Keywords

Genome engineering TALE-based nucleases TALENs Homodimeric TALENs Saccharomyces cerevisiae Targeted genome modification 

Notes

Acknowledgments

We thank the Bioscience Core Facility and Optical, Imaging Core Lab King Abdullah University of Science and Technology KAUST for technical assistance. We also thank Nina Fedoroff and the members of the genome engineering group at KAUST for their helpful discussions and technical assistance throughout the preparation of the manuscript. No conflict of interest declared. This research is funded from the Center for Desert Agriculture and genome engineering group baseline funding.

Supplementary material

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References

  1. Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I (1998) FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci USA 95:10570–10575PubMedCrossRefPubMedCentralGoogle Scholar
  2. Boch J, Bonas U (2010) Xanthomonas AvrBs3 Family-Type III Effectors: discovery and function. Annu Rev Phytopathol 48:419–436PubMedCrossRefGoogle Scholar
  3. 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
  4. Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846PubMedCrossRefGoogle Scholar
  5. Bogdanove AJ, Schornack S, Lahaye T (2010) TAL effectors: finding plant genes for disease and defense. Curr Opin Plant Biol 13:394–401PubMedCrossRefGoogle Scholar
  6. 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:e82PubMedCrossRefPubMedCentralGoogle Scholar
  7. 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 186:757–761PubMedCrossRefPubMedCentralGoogle Scholar
  8. Clark KJ, Voytas DF, Ekker SC (2011) A TALE of two nucleases: gene targeting for the masses? Zebrafish 8:147–149PubMedCrossRefPubMedCentralGoogle Scholar
  9. Cong L, Zhou R, Y-c Kuo, Cunniff M, Zhang F (2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun 3:968PubMedCrossRefPubMedCentralGoogle Scholar
  10. Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, Reyon D, Dahlborg EJ, Goodwin MJ, Coffman AP, Dobbs D, Joung JK, Voytas DF, Stupar RM (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol 156:466–473PubMedCrossRefPubMedCentralGoogle Scholar
  11. Dahlem TJ, Hoshijima K, Jurynec MJ, Gunther D, Starker CG, Locke AS, Weis AM, Voytas DF, Grunwald DJ (2012) Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet 8:e1002861PubMedCrossRefPubMedCentralGoogle Scholar
  12. Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu JK, Shi Y, Yan N (2012a) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335:720–723PubMedCrossRefPubMedCentralGoogle Scholar
  13. Deng D, Yin P, Yan C, Pan X, Gong X, Qi S, Xie T, Mahfouz M, Zhu JK, Yan N, Shi Y (2012b) Recognition of methylated DNA by TAL effectors. Cell Res 22:1502–1504PubMedCrossRefPubMedCentralGoogle Scholar
  14. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26:702–708PubMedCrossRefPubMedCentralGoogle Scholar
  15. Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425PubMedCrossRefPubMedCentralGoogle Scholar
  16. Guo J, Gaj T, Barbas Iii CF (2010) Directed evolution of an enhanced and highly efficient fokI cleavage domain for zinc finger nucleases. J Mol Biol 400:96–107PubMedCrossRefPubMedCentralGoogle Scholar
  17. Guthrie C, Fink GR (1991) Guide to yeast genetics and molecular biology. Methods Enzymol 3–37 Google Scholar
  18. Heuer H, Yin YN, Xue QY, Smalla K, Guo JH (2007) Repeat domain diversity of avrBs3-like genes in Ralstonia solanacearum strains and association with host preferences in the field. Appl Environ Microbiol 73:4379–4384PubMedCrossRefPubMedCentralGoogle Scholar
  19. 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
  20. Hutter KJ, Eipel HE (1979) Microbial determinations by flow cytometry. J Gen Microbiol 113:369–375Google Scholar
  21. Kay S, Boch J, Bonas U (2005) Characterization of AvrBs3-like effectors from a Brassicaceae pathogen reveals virulence and avirulence activities and a protein with a novel repeat architecture. Mol Plant-Microbe Interact 18:838–848PubMedCrossRefGoogle Scholar
  22. Leduc A, He CH, Ramotar D (2003) Disruption of the Saccharomyces cerevisiae cell-wall pathway gene SLG1 causes hypersensitivity to the antitumor drug bleomycin. Mol Genet Genomics 269:78–89PubMedGoogle Scholar
  23. Li L, Piatek MJ, Atef A, Piatek A, Wibowo A, Fang X, Sabir JS, Zhu JK, Mahfouz MM (2012a) Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification. Plant Mol Biol 78:407–416PubMedCrossRefPubMedCentralGoogle Scholar
  24. Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012b) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotechnol 30:390–392PubMedCrossRefGoogle Scholar
  25. Li L, Atef A, Piatek A, Ali Z, Piatek M, Aouida M, Sharakou A, Mahjoub A, Wang G, Khan S, Fedoroff NV, Zhu JK, Mahfouz M (2013) Characterization and DNA-binding specificities of Ralstonia TAL-like effectors. Mol Plant 6:1318–1330PubMedCrossRefGoogle Scholar
  26. Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B, Deng W-M, Jiao R (2012) Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J Genet Genom 39:209–215CrossRefGoogle Scholar
  27. Mahfouz MM (2010) RNA-directed DNA methylation: mechanisms and functions. Plant Signal Behav 5:806–816PubMedCrossRefPubMedCentralGoogle Scholar
  28. Mahfouz MM, Li L (2011) TALE nucleases and next generation GM crops. GM Crop 2:99–103CrossRefGoogle Scholar
  29. Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A, Fang X, Zhu JK (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108:2623–2628PubMedCrossRefPubMedCentralGoogle Scholar
  30. Mahfouz MM, Li L, Piatek M, Fang X, Mansour H, Bangarusamy DK, Zhu JK (2012) Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein. Plant Mol Biol 3:311–321CrossRefGoogle Scholar
  31. Mak AN, Bradley P, Cernadas RA, Bogdanove AJ, Stoddard BL (2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335:716–719PubMedCrossRefPubMedCentralGoogle Scholar
  32. Masson JY, Ramotar D (1997) Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III. Mol Microbiol 24:711–721PubMedCrossRefGoogle Scholar
  33. 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 107:21617–21622PubMedCrossRefPubMedCentralGoogle Scholar
  34. Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326:1501PubMedCrossRefGoogle Scholar
  35. Ramirez CL, Foley JE, Wright DA, Muller-Lerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA, Cathomen T, Voytas DF, Joung JK (2008) Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5:374–375PubMedCrossRefGoogle Scholar
  36. Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30:460–465PubMedCrossRefPubMedCentralGoogle Scholar
  37. Romer P, Hahn S, Jordan T, Strauss T, Bonas U, Lahaye T (2007) Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 318:645–648PubMedCrossRefGoogle Scholar
  38. 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 29:697–698PubMedCrossRefPubMedCentralGoogle Scholar
  39. Shan Q, Wang Y, Chen K, Liang Z, Li JUN, Zhang Y, Zhang K, Liu J, Voytas DF, Zheng X, Zhang Y, Gao C (2013) Rapid and efficient gene modification in rice and Brachypodium using TALENs. Molecular Plant 4:1365–1368CrossRefGoogle Scholar
  40. Sherman F, Fink GR, Hicks J (1983) Laboratory course manual for methods in yeast genetics.  Cold Spring Harbor Laboratory, Plainview, NY Google Scholar
  41. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437–441PubMedCrossRefGoogle Scholar
  42. Streubel J, Blucher C, Landgraf A, Boch J (2012) TAL effector RVD specificities and efficiencies. Nat Biotechnol 30:593–595PubMedCrossRefGoogle Scholar
  43. Voytas DF, Joung JK (2009) Plant science. DNA binding made easy. Science 326:1491–1492PubMedCrossRefGoogle Scholar
  44. Wah DA, Bitinaite J, Schildkraut I, Aggarwal AK (1998) Structure of FokI has implications for DNA cleavage. Proc Natl Acad Sci USA 95:10564–10569PubMedCrossRefPubMedCentralGoogle Scholar
  45. Zhang F, Voytas DF (2011) Targeted mutagenesis in Arabidopsis using zinc-finger nucleases. Methods Mol Biol 701:167–177PubMedCrossRefGoogle Scholar
  46. Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161:20–27PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Mustapha Aouida
    • 1
  • Marek J. Piatek
    • 1
  • Dhinoth K. Bangarusamy
    • 2
  • Magdy M. Mahfouz
    • 1
    Email author
  1. 1.Center for Desert Agriculture and Division of Biological SciencesKing Abdullah University of Science and TechnologyThuwalKingdom of Saudi Arabia
  2. 2.Bioscience Core LabKing Abdullah University of Science and TechnologyThuwalKingdom of Saudi Arabia

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