Transgenic Expression and Genome Editing by Electroporation of Zebrafish Embryos

Mar Biotechnol (NY). 2020 Oct;22(5):644-650. doi: 10.1007/s10126-020-09985-0. Epub 2020 Aug 4.

Abstract

Microinjection is predominantly used to produce genetically modified fish. However, there are certain difficulties involved in some fish species. In this study, we tested an alternative method to produce genetically modified zebrafish by delivering DNA and other materials into embryos by electroporation. We optimized the electroporation conditions of a square wave electroporation system that work efficiently for the introduction of plasmid DNA, recombinant Cas9 nuclease and synthetic dual guide RNAs. Transgenic expression was observed in a wide range of tissues, which is comparable with those obtained by microinjection. We further determined that efficient gene delivery can be achieved during the cleavage stage. This study describes detailed electroporation parameters for gene delivery with high efficiency and low toxicity, providing a novel method to generate transgenic lines and genome editing.

Keywords: CRISPR/Cas9; Electroporation; Genome editing; Transgenesis; Zebrafish embryos.

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • DNA / genetics
  • Electroporation / methods*
  • Embryo, Nonmammalian
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Editing / methods*
  • Gene Transfer Techniques*
  • Plasmids / genetics
  • RNA, Guide / genetics
  • Zebrafish / embryology
  • Zebrafish / genetics*

Substances

  • RNA, Guide
  • DNA
  • Endonucleases