Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Efficient genome engineering approaches for the short-lived African turquoise killifish

Nature Protocols volume 11, pages 2010–2028 (2016)Cite this article

Subjects

Abstract

A central challenge in experimental aging research is the lack of short-lived vertebrate models for genetic studies. Here we present a comprehensive protocol for efficient genome engineering in the African turquoise killifish (Nothobranchius furzeri), which is the shortest-lived vertebrate in captivity with a median life span of 4–6 months. By taking advantage of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system and the turquoise killifish genome, this platform enables the generation of knockout alleles via nonhomologous end joining (NHEJ) and knock-in alleles via homology-directed repair (HDR). We include guidelines for guide RNA (gRNA) target design, embryo injection and hatching, germ-line transmission and for minimizing off-target effects. We also provide strategies for Tol2-based transgenesis and large-scale husbandry conditions that are critical for success. Because of the fast life cycle of the turquoise killifish, stable lines can be generated as rapidly as 2–3 months, which is much faster than other fish models. This protocol provides powerful genetic tools for studying vertebrate aging and aging-related diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The turquoise killifish model for experimental aging research.
Figure 2: CRISPR/Cas9-based genome editing.
Figure 3: Tol2-based transgenesis.
Figure 4: Injection and hatching.
Figure 5: Preparation of injection needles.
Figure 6: Genome engineering in the turquoise killifish.

Similar content being viewed by others

References

  1. Harel, I. & Brunet, A. The African turquoise killifish: a model for exploring vertebrate aging and diseases in the fast lane. Cold Spring Harb. Symp. Quant. Biol. 80, 275–279 (2015).

    Article  PubMed  Google Scholar 

  2. Harel, I. et al. A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate. Cell 160, 1013–1026 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cellerino, A., Valenzano, D.R. & Reichard, M. From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology. Biol. Rev. Camb. Philos. Soc. 91, 511–533 (2015).

    Article  PubMed  Google Scholar 

  4. Genade, T. et al. Annual fishes of the genus Nothobranchius as a model system for aging research. Aging Cell 4, 223–233 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Scheel, J.J. Atlas of killifishes of the Old World (T.F.H. Publications, 1990).

  6. Valenzano, D.R. et al. The African turquoise killifish genome provides insights into evolution and genetic architecture of lifespan. Cell 163, 1539–1554 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tozzini, E.T. et al. Parallel evolution of senescence in annual fishes in response to extrinsic mortality. BMC Evol. Biol. 13, 77 (2013).

    Article  PubMed  Google Scholar 

  8. Hartmann, N. et al. Mitochondrial DNA copy number and function decrease with age in the short-lived fish Nothobranchius furzeri. Aging Cell 10, 824–831 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Valenzano, D.R., Terzibasi, E., Cattaneo, A., Domenici, L. & Cellerino, A. Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri. Aging Cell 5, 275–278 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Terzibasi, E. et al. Large differences in aging phenotype between strains of the short-lived annual fish Nothobranchius furzeri. PloS One 3, e3866 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Valenzano, D.R. et al. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296–300 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Terzibasi, E. et al. Effects of dietary restriction on mortality and age-related phenotypes in the short-lived fish Nothobranchius furzeri. Aging Cell 8, 88–99 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Schartl, M. Beyond the zebrafish: diverse fish species for modeling human disease. Dis. Mod. Mech. 7, 181–192 (2014).

    Google Scholar 

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

  15. Gomes, N.M., Shay, J.W. & Wright, W.E. Telomere biology in metazoa. FEBS Lett. 584, 3741–3751 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gemberling, M., Bailey, T.J., Hyde, D.R. & Poss, K.D. The zebrafish as a model for complex tissue regeneration. Trends Genet. 29, 611–620 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Santoriello, C. & Zon, L.I. Hooked! Modeling human disease in zebrafish. J. Clin. Invest. 122, 2337–2343 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Jones, F.C. et al. The genomic basis of adaptive evolution in threespine sticklebacks. Nature 484, 55–61 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yamamoto, Y., Stock, D.W. & Jeffery, W.R. Hedgehog signalling controls eye degeneration in blind cavefish. Nature 431, 844–847 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Valenzano, D.R. et al. Mapping loci associated with tail color and sex determination in the short-lived fish Nothobranchius furzeri. Genetics 183, 1385–1395 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Reichwald, K. et al. Insights into sex chromosome evolution and aging from the genome of a short-lived fish. Cell 163, 1527–1538 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Podrabsky, J.E. & Hand, S.C. The bioenergetics of embryonic diapause in an annual killifish, Austrofundulus limnaeus. J. Exp. Biol. 202 (Part 19), 2567–2580 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Wourms, J.P. The developmental biology of annual fishes. 3. Pre-embryonic and embryonic diapause of variable duration in the eggs of annual fishes. J. Exp. Zool. 182, 389–414 (1972).

    Article  CAS  PubMed  Google Scholar 

  24. Valdesalici, S. & Cellerino, A. Extremely short lifespan in the annual fish Nothobranchius furzeri. Proc. Biol. Sci. 270 (Suppl. 2), S189–S191 (2003).

    PubMed  PubMed Central  Google Scholar 

  25. Kim, Y., Nam, H.G. & Valenzano, D.R. The short-lived African turquoise killifish: an emerging experimental model for ageing. Dis. Mod. Mech. 9, 115–129 (2016).

    Article  Google Scholar 

  26. Reichwald, K. et al. High tandem repeat content in the genome of the short-lived annual fish Nothobranchius furzeri: a new vertebrate model for aging research. Genome Biol. 10, R16 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Kirschner, J. et al. Mapping of quantitative trait loci controlling lifespan in the short-lived fish Nothobranchius furzeri--a new vertebrate model for age research. Aging Cell 11, 252–261 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Valenzano, D.R., Sharp, S. & Brunet, A. Transposon-mediated transgenesis in the short-lived African killifish Nothobranchius furzeri, a vertebrate model for aging. G3 (Bethesda) 1, 531–538 (2011).

    Article  CAS  Google Scholar 

  29. Hartmann, N. & Englert, C. A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri). Dev. Dyn. 241, 1133–1141 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Allard, J.B., Kamei, H. & Duan, C. Inducible transgenic expression in the short-lived fish Nothobranchius furzeri. J. Fish Biol. 82, 1733–1738 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Doudna, J.A. & Sontheimer, E.J. Methods in enzymology. The use of CRISPR/Cas9, ZFNs, and TALENs in generating site-specific genome alterations. Preface. Methods Enzymol. 546, xix–xx (2014).

    Article  PubMed  Google Scholar 

  36. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Heidenreich, M. & Zhang, F. Applications of CRISPR-Cas systems in neuroscience. Nat. Rev. Neurosci. 17, 36–44 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. 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 

  39. Davidson, A.E. et al. Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev. Biol. 263, 191–202 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Kawakami, K., Shima, A. & Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc. Natl. Acad. Sci. USA 97, 11403–11408 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Grabher, C. & Wittbrodt, J. Meganuclease and transposon mediated transgenesis in medaka. Genome Biol. 8 (Suppl. 1), S10 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Bedell, V.M. et al. In vivo genome editing using high-efficiency TALEN system. Nature 491, 114–118 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Chang, N. et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23, 465–472 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Auer, T.O., Duroure, K., De Cian, A., Concordet, J.P. & Del Bene, F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 24, 142–153 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jao, L.E., Wente, S.R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl. Acad. Sci. USA 110, 13904–13909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Polačik, M., Blažek, R. & Reichard, M. Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri. Nat. Protoc. 11, 1396–1413 (2016).

    Article  PubMed  CAS  Google Scholar 

  48. Blazek, R., Polacik, M. & Reichard, M. Rapid growth, early maturation and short generation time in African annual fishes. EvoDevo 4, 24 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Montague, T.G., Cruz, J.M., Gagnon, J.A., Church, G.M. & Valen, E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401–W407 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Labun, K., Montague, T.G., Gagnon, J.A., Thyme, S.B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44, W272–W276 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gagnon, J.A. et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One 9, e98186 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Thomas, H.R., Percival, S.M., Yoder, B.K. & Parant, J.M. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis. PLoS One 9, e114632 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Carrington, B., Varshney, G.K., Burgess, S.M. & Sood, R. CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity. Nucleic Acids Res. 43, e157 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Shalem, O., Sanjana, N.E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Scheer, N. & Campos-Ortega, J.A. Use of the Gal4-UAS technique for targeted gene expression in the zebrafish. Mech. Dev. 80, 153–158 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Dong, J. & Stuart, G.W. Transgene manipulation in zebrafish by using recombinases. Methods Cell Biol. 77, 363–379 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Pan, X., Wan, H., Chia, W., Tong, Y. & Gong, Z. Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system. Transgenic Res. 14, 217–223 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Langenau, D.M. et al. Cre/lox-regulated transgenic zebrafish model with conditional myc-induced T cell acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 102, 6068–6073 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Huang, C.J. et al. Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines. Dev. Dyn. 233, 1294–1303 (2005).

    Article  CAS  PubMed  Google Scholar 

  61. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Larson, M.H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Dominguez, A.A., Lim, W.A. & Qi, L.S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016).

    Article  CAS  PubMed  Google Scholar 

  65. Chari, R., Mali, P., Moosburner, M. & Church, G.M. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat. Methods 12, 823–826 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Kim, D., Kim, S., Kim, S., Park, J. & Kim, J.S. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 26, 406–415 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Kleinstiver, B.P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M. & Altman, D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Yamamoto, T.-o. Changes of the cortical layer of the egg of Oryzias latipes at the time of fertilization. Proc. Imp. Acad. Tokyo 15, 269–271 (1939).

    Article  Google Scholar 

  71. Dolfi, L., Ripa, R. & Cellerino, A. Transition to annual life history coincides with reduction in cell cycle speed during early cleavage in three independent clades of annual killifish. EvoDevo 5, 32 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Podrabsky, J.E. Husbandry of the annual killifish Austrofundulus limnaeus with special emphasis on the collection and rearing of embryos. Environ. Biol. Fishes 54, 421–431 (1999).

    Article  Google Scholar 

  73. Clark, K.J., Urban, M.D., Skuster, K.J. & Ekker, S.C. Transgenic zebrafish using transposable elements. Methods Cell Biol. 104, 137–149 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Kawakami, K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol. 8 (Suppl. 1), S7 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Kwan, K.M. et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007).

    Article  CAS  PubMed  Google Scholar 

  76. Huang, C.J., Tu, C.T., Hsiao, C.D., Hsieh, F.J. & Tsai, H.J. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn. 228, 30–40 (2003).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank all members of the Brunet lab, specifically B.A. Benayoun and P.P. Singh for all the work involving the genome project. We are grateful to our coauthors on Harel et al. (2015). We thank A.S. McKay for generating the files for 3D printing, C.-K. Hu for taking the photos for Figure 3b, A.S. McKay, C.-K. Hu, L. Booth, Y. Kim, W. Wei, P. Abitua and M. Concha for critical reading of the manuscript and important suggestions, and S. Thyme, M. Reichard and M.L. Powell for critical scientific discussions. This work was supported by US National Institutes of Health (NIH) DP1AG044848 and the Glenn Laboratories for the Biology of Aging (A.B.), by the Max Planck Society and the Max Planck Institute for Biology of Ageing (D.R.V.) and by the Damon Runyon, Rothschild and HFSP fellowships (I.H.).

Author information

Authors and Affiliations

  1. Department of Genetics, Stanford University, Stanford, California, USA

    Itamar Harel & Anne Brunet

  2. Max Planck Institute for Biology of Ageing, Cologne, Germany

    Dario Riccardo Valenzano

  3. CECAD, University of Cologne, Cologne, Germany

    Dario Riccardo Valenzano

  4. Glenn Laboratories for the Biology of Aging at Stanford, Stanford, California, USA

    Anne Brunet

Authors
  1. Itamar Harel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dario Riccardo Valenzano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne Brunet
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

I.H. and A.B. conceived the project, and I.H. performed the experiments. I.H. and A.B. wrote the manuscript, with assistance from D.R.V.

Corresponding authors

Correspondence to Itamar Harel or Anne Brunet.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Injection mold 3D-printing design. 3D-printing design for the injection mold with optimized dimensions. The design is provided in two commonly used file formats for 3D printing (.dwg and .stl), allowing for better flexibility in regard to the requirements of 3D-printing service providers. The .dwg format can be used by many programs, including DraftSight, AutoCAD, IntelliCAD and Caddie. The .stl format can be used by many programs, including AutoCAD, CloudCompare and FreeCAD. (ZIP 39 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Harel, I., Valenzano, D. & Brunet, A. Efficient genome engineering approaches for the short-lived African turquoise killifish. Nat Protoc 11, 2010–2028 (2016). https://doi.org/10.1038/nprot.2016.103

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.103

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing