Targeted mutagenesis in zebrafish using customized zinc-finger nucleases

Nat Protoc. 2009;4(12):1855-67. doi: 10.1038/nprot.2009.209.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Deoxyribonucleases / chemistry
  • Deoxyribonucleases / metabolism*
  • Embryo, Nonmammalian
  • Frameshift Mutation*
  • Genetic Vectors
  • Mutagenesis, Site-Directed / methods*
  • Polymerase Chain Reaction
  • Protein Engineering
  • Restriction Mapping
  • Zebrafish / embryology
  • Zebrafish / genetics*
  • Zinc Fingers*


  • Deoxyribonucleases