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Targeted mutagenesis in zebrafish using customized zinc-finger nucleases

Nature Protocols volume 4, pages 1855–1868 (2009)Cite this article

Abstract

Zebrafish mutants have traditionally been obtained by using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc-finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc-Finger Consortium reagents for constructing engineered zinc-finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within 4 months.

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Figure 1: A diagram of zinc-finger nucleases (ZFN)-induced insertion or deletion (indel) mutations.
Figure 2
Figure 3: A representative gel of in vitro transcribed RNAs encoding zinc-finger nucleases (ZFNs).
Figure 4
Figure 5
Figure 6: Representative results of fluorescent PCR analysis of (a) a pool of wild-type embryos and (b) a pool of embryos from a founder.

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References

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

    Article  CAS  PubMed  Google Scholar 

  2. Porteus, M.H. & Carroll, D. Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–973 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M.H. & Chandrasegaran, S. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 33, 5978–5990 (2005).

    Article  CAS  PubMed  PubMed Central  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. Wu, J., Kandavelou, K. & Chandrasegaran, S. Custom-designed zinc finger nucleases: what is next? Cell. Mol. Life Sci. 64, 2933–2944 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 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 

  7. 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  Google Scholar 

  8. Kim, Y.G., Shi, Y., Berg, J.M. & Chandrasegaran, S. Site-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions. Gene 203, 43–49 (1997).

    Article  CAS  PubMed  Google Scholar 

  9. 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 

  10. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. 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 

  12. Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. 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 

  14. 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 

  15. 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 

  16. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702–708 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Foley, J.E. et al. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). PloS. ONE 4, e4348 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D. & Wolfe, S.A. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695–701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Beumer, K.J. et al. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 19821–19826 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J.K. & Carroll, D. Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172, 2391–2403 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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  Google Scholar 

  22. 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 

  23. Moehle, E.A. et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 104, 3055–3060 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. 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 

  25. Shukla, V.K. et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437–441 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. 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 

  27. Cai, C.Q. et al. Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol. Biol. 69, 699–709 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Hurt, J.A., Thibodeau, S.A., Hirsh, A.S., Pabo, C.O. & Joung, J.K. Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection. Proc. Natl. Acad. Sci. USA 100, 12271–12276 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Cornu, T.I. et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Smith, J., Bibikova, M., Whitby, F.G., Reddy, A.R., Chandrasegaran, S. & Carroll, D. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 28, 3361–3369 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mani, M., Smith, J., Kandavelou, K., Berg, J.M. & Chandrasegaran, S. Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem. Biophys. Res. Commun. 334, 1191–1197 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Beerli, R.R. & Barbas III, C.F. Engineering polydactyl zinc-finger transcription factors. Nat. Biotechnol. 20, 135–141 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Segal, D.J. The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment. Methods 26, 76–83 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Liu, Q., Xia, Z., Zhong, X. & Case, C.C. Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J. Biol. Chem. 277, 3850–3856 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Bae, K.H. et al. Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat. Biotechnol. 21, 275–280 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. 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 

  37. Ramirez, C.L. et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat. Methods 5, 374–375 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Carroll, D., Morton, J.J., Beumer, K.J. & Segal, D.J. Design, construction and in vitro testing of zinc finger nucleases. Nat. Protoc. 1, 1329–1341 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Wright, D.A. et al. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat. Protoc. 1, 1637–1652 (2006).

    Article  PubMed  Google Scholar 

  41. Sander, J.D., Zaback, P., Joung, J.K., Voytas, D.F. & Dobbs, D. Zinc finger targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res. 35, W599–W605 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Szczepek, M., Brondani, V., Buchel, J., Serrano, L., Segal, D.J. & Cathomen, T. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat. Biotechnol. 25, 786–793 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Rembold, M., Lahiri, K., Foulkes, N.S. & Wittbrodt, J. Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct. Nat. Protoc. 1, 1133–1139 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio) (The University of Oregon Press, Eugene, Oregon, 2000).

  45. Yuan, S. & Sun, Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J. Vis. Exp. published online, doi:10.3791/1113 (7 May 2009).

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Acknowledgements

We thank Dr. A.J. Giraldez for valuable suggestions and D. Cotelle for technical help on the fluorescent PCR analysis, Drs. P. Schlueter and C. Sachidanandan for helpful suggestions on the manuscript. J.E.F., M.L.M. and J.K.J. are supported by the NIH (R01GM069906, R21RR024189, and R21HL091808) and the MGH Pathology Service. R.T.P. and J.-R.J.Y. are supported by the NIH (CA118498 and GM88040) and the Ned Sahin Fund. J.-R.J.Y. is also supported by the NIH (AG031300) and the Claflin Distinguished Scholar Award.

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Authors and Affiliations

  1. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Jonathan E Foley, Morgan L Maeder & J Keith Joung

  2. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jonathan E Foley, Morgan L Maeder & J Keith Joung

  3. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Joseph Pearlberg

  5. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    J Keith Joung

  6. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Randall T Peterson & Jing-Ruey J Yeh

  7. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Randall T Peterson & Jing-Ruey J Yeh

  8. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Randall T Peterson

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  1. Jonathan E Foley
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Contributions

J.P. provided the protocol for plasmid DNA isolation in a 96-well format. All the other authors contributed extensively to the protocol development and preparation of the manuscript.

Corresponding author

Correspondence to Jing-Ruey J Yeh.

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Competing interests

J.K.J. is an inventor on patent applications which describe the OPEN zinc finger engineering method. All other authors declare that they have no competing financial interests.

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Foley, J., Maeder, M., Pearlberg, J. et al. Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nat Protoc 4, 1855–1868 (2009). https://doi.org/10.1038/nprot.2009.209

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