Protocol

CRISPR

Volume 1311 of the series Methods in Molecular Biology pp 317-334

Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases

  • Woong Y. HwangAffiliated withCardiovascular Research Center, Massachusetts General HospitalDepartment of Medicine, Harvard Medical School
  • , Yanfang FuAffiliated withMolecular Pathology Unit, Center for Cancer Research, Massachusetts General HospitalCenter for Computational and Integrative Biology, Massachusetts General HospitalDepartment of Pathology, Harvard Medical School
  • , Deepak ReyonAffiliated withMolecular Pathology Unit, Center for Cancer Research, Massachusetts General HospitalCenter for Computational and Integrative Biology, Massachusetts General HospitalDepartment of Pathology, Harvard Medical School
  • , Andrew P. W. GonzalesAffiliated withCardiovascular Research Center, Massachusetts General HospitalDepartment of Medicine, Harvard Medical School
  • , J. Keith JoungAffiliated withMolecular Pathology Unit, Center for Cancer Research, Massachusetts General HospitalCenter for Computational and Integrative Biology, Massachusetts General HospitalDepartment of Pathology, Harvard Medical School Email author 
  • , Jing-Ruey Joanna YehAffiliated withCardiovascular Research Center, Massachusetts General HospitalDepartment of Medicine, Harvard Medical School

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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.

Key words

CRISPR Cas9 Gene-editing Genome engineering Zebrafish T7E1