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Construction and Application of Site-Specific Artificial Nucleases for Targeted Gene Editing

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Gene Function Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1101))

Abstract

Artificial nucleases have developed into powerful tools for introducing precise genome modifications in a wide variety of species. In this chapter the authors provide detailed protocols for rapidly constructing zinc finger nucleases (ZFNs) and TALE nucleases (TALENs) and evaluating their activity for the targeted generation of InDels within the zebrafish genome.

Fatma O. Kok and Ankit Gupta contributed equally to this work.

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References

  1. Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188(4):773–782. doi:10.1534/genetics.111.131433

    Article  PubMed  CAS  Google Scholar 

  2. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25(7):786–793. doi:10.1038/nbt1317

    Article  PubMed  CAS  Google Scholar 

  3. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25(7):778–785. doi:10.1038/nbt1319

    Article  PubMed  CAS  Google Scholar 

  4. Doyon Y, Vo TD, Mendel MC, Greenberg SG, Wang J, Xia DF, Miller JC, Urnov FD, Gregory PD, Holmes MC (2011) Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8(1):74–79. doi:10.1038/nmeth.1539

    Article  PubMed  CAS  Google Scholar 

  5. Zhu C, Smith T, McNulty J, Rayla AL, Lakshmanan A, Siekmann AF, Buffardi M, Meng X, Shin J, Padmanabhan A, Cifuentes D, Giraldez AJ, Look AT, Epstein JA, Lawson ND, Wolfe SA (2011) Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish. Development 138(20):4555–4564. doi:10.1242/dev.066779

    Article  PubMed  CAS  Google Scholar 

  6. Gupta A, Christensen RG, Rayla AL, Lakshmanan A, Stormo GD, Wolfe SA (2012) An optimized two-finger archive for ZFN-mediated gene targeting. Nat Methods 9(6):588–590. doi:10.1038/nmeth.1994

    Article  PubMed  CAS  Google 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. Nucl Acid Res 39(12):e82. doi:10.1093/nar/gkr218

    Article  CAS  Google Scholar 

  8. Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29(8):699–700. doi:10.1038/nbt.1939

    Article  PubMed  Google Scholar 

  9. 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(8):697–698. doi:10.1038/nbt.1934

    Article  PubMed  CAS  Google Scholar 

  10. Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L, Gregory PD, Anegon I, Cost GJ (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29(8):695–696. doi:10.1038/nbt.1940

    Article  PubMed  CAS  Google Scholar 

  11. 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(5):460–465. doi:10.1038/nbt.2170

    Article  PubMed  CAS  Google Scholar 

  12. Weber E, Gruetzner R, Werner S, Engler C, Marillonnet S (2011) Assembly of designer TAL effectors by golden gate cloning. PLoS ONE 6(5):e19722. doi:10.1371/journal.pone.0019722

    Article  PubMed  CAS  Google Scholar 

  13. Engler C, Marillonnet S (2011) Generation of families of construct variants using golden gate shuffling. Methods Mol Biol 729:167–181. doi:10.1007/978-1-61779-065-2_11

    Article  PubMed  Google Scholar 

  14. Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3(11):e3647. doi:10.1371/journal.pone.0003647

    Article  PubMed  Google Scholar 

  15. Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4(5):e5553. doi:10.1371/journal.pone.0005553

    Article  PubMed  Google Scholar 

  16. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148. doi:10.1038/nbt.1755

    Article  PubMed  CAS  Google Scholar 

  17. Kay S, Bonas U (2009) How Xanthomonas type III effectors manipulate the host plant. Curr Opin Microbiol 12(1):37–43. doi:10.1016/j.mib.2008.12.006

    Article  PubMed  CAS  Google Scholar 

  18. Boch J, Bonas U (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol 48:419–436. doi:10.1146/annurev-phyto-080508-081936

    Article  PubMed  CAS  Google Scholar 

  19. Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326(5959):1501. doi:10.1126/science.1178817

    Article  PubMed  CAS  Google Scholar 

  20. 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(5959):1509–1512. doi:10.1126/science.1178811

    Article  PubMed  CAS  Google Scholar 

  21. Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu JK, Shi Y, Yan N (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335(6069):720–723. doi:10.1126/science.1215670

    Article  PubMed  CAS  Google Scholar 

  22. 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(6069):716–719. doi:10.1126/science.1216211

    Article  PubMed  CAS  Google Scholar 

  23. Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK, Bogdanove AJ (2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucl Acid Res 40(Web Server issue):W117–W122. doi:10.1093/nar/gks608

    Article  CAS  Google Scholar 

  24. 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. Nucl Acid Res 39(21):9283–9293. doi:10.1093/nar/gkr597

    Article  CAS  Google Scholar 

  25. 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(6):2623–2628. doi:10.1073/pnas.1019533108

    Article  PubMed  CAS  Google Scholar 

  26. Christian ML, Demorest ZL, Starker CG, Osborn MJ, Nyquist MD, Zhang Y, Carlson DF, Bradley P, Bogdanove AJ, Voytas DF (2012) Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues. PLoS ONE 7(9):e45383. doi:10.1371/journal.pone.0045383

    Article  PubMed  CAS  Google Scholar 

  27. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734. doi:10.1038/nbt.1927

    Article  PubMed  CAS  Google Scholar 

  28. Wood AJ, Lo TW, Zeitler B, Pickle CS, Ralston EJ, Lee AH, Amora R, Miller JC, Leung E, Meng X, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Meyer BJ (2011) Targeted genome editing across species using ZFNs and TALENs. Science 333(6040):307. doi:10.1126/science.1207773

    Article  PubMed  CAS  Google Scholar 

  29. Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug Ii RG, Tan W, Penheiter SG, Ma AC, Leung AY, Fahrenkrug SC, Carlson DF, Voytas DF, Clark KJ, Essner JJ, Ekker SC (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491:114–118. doi:10.1038/nature11537

    Article  PubMed  CAS  Google Scholar 

  30. Rosen JN, Sweeney MF, Mably JD (2009) Microinjection of zebrafish embryos to analyze gene function. J Vis Exp (25): 1115, DOI:10.3791/1115

  31. Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26(6):695–701. doi:10.1038/nbt1398

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Program in Gene Function & Expression, Department of Biochemistry & Molecular Pharmacology, UMass Medical School, Worcester, MA, USA

    Fatma O. Kok, Ankit Gupta, Nathan D. Lawson & Scot A. Wolfe

Authors
  1. Fatma O. Kok
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  2. Ankit Gupta
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  3. Nathan D. Lawson
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  4. Scot A. Wolfe
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Editor information

Editors and Affiliations

  1. Dept of Mathematics & Statistics, The College of New Jersey, Ewing, New Jersey, USA

    Michael F. Ochs

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Kok, F.O., Gupta, A., Lawson, N.D., Wolfe, S.A. (2014). Construction and Application of Site-Specific Artificial Nucleases for Targeted Gene Editing. In: Ochs, M. (eds) Gene Function Analysis. Methods in Molecular Biology, vol 1101. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-721-1_13

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  • DOI: https://doi.org/10.1007/978-1-62703-721-1_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-720-4

  • Online ISBN: 978-1-62703-721-1

  • eBook Packages: Springer Protocols

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