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Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases

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CRISPR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1311))

Abstract

In recent years, the zebrafish has become a critical contributor to various areas of biomedical research, advancing our fundamental understanding of biomedicine and helping discover candidate therapeutics for human diseases. Nevertheless, to further extend the power of this important model organism requires a robust and simple-to-use genome editing platform that will enable targeted gene knockouts and introduction of specific mutations identified in human diseases into the zebrafish genome. We describe here protocols for creating insertion or deletion (indel) mutations or precise sequence modifications in zebrafish genes using customizable CRISPR-Cas9 RNA-guided nucleases (RGNs). These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms.

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Acknowledgements

The authors would like to thank Drs. Randall Peterson, Morgan Maeder, and Jeffry Sander for their contributions on various aspects of this study. W.Y.H., A.P.W.G., and J.-R.J.Y. are supported by the National Institutes of Health (R01 GM088040, K01 AG031300, and R01 CA140188) and by the MGH Claflin Distinguished Scholar Award. Y.F., D.R., and J.K.J. are supported by the National Institutes of Health (DP1 GM105378 and R01 GM088040) and by The Jim and Ann Orr MGH Research Scholar Award.

Conflict of Interest Declaration: J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies.

Author information

Authors and Affiliations

  1. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, 02129, USA

    Woong Y. Hwang, Andrew P. W. Gonzales & Jing-Ruey Joanna Yeh

  2. Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA

    Woong Y. Hwang, Andrew P. W. Gonzales & Jing-Ruey Joanna Yeh

  3. Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, 02129, USA

    Yanfang Fu, Deepak Reyon & J. Keith Joung

  4. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, 02129, USA

    Yanfang Fu, Deepak Reyon & J. Keith Joung

  5. Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA

    Yanfang Fu, Deepak Reyon & J. Keith Joung

Authors
  1. Woong Y. Hwang
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  2. Yanfang Fu
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  3. Deepak Reyon
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  4. Andrew P. W. Gonzales
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  5. J. Keith Joung
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  6. Jing-Ruey Joanna Yeh
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Corresponding author

Correspondence to J. Keith Joung .

Editor information

Editors and Affiliations

  1. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Magnus Lundgren

  2. Department of Molecular Biology MIMS, Umeå University, Umeå, Sweden

    Emmanuelle Charpentier

  3. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

    Peter C. Fineran

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Hwang, W.Y., Fu, Y., Reyon, D., Gonzales, A.P.W., Joung, J.K., Yeh, JR.J. (2015). Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_21

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  • DOI: https://doi.org/10.1007/978-1-4939-2687-9_21

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2686-2

  • Online ISBN: 978-1-4939-2687-9

  • eBook Packages: Springer Protocols

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