Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos

Cell Res. 2013 Apr;23(4):465-72. doi: 10.1038/cr.2013.45. Epub 2013 Mar 26.

Abstract

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp(y11) mutant embryos or cardia bifida phenotypes in fau(tm236a) mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Embryo, Nonmammalian
  • Fish Proteins / genetics*
  • Fish Proteins / metabolism
  • GATA Transcription Factors / genetics
  • GATA Transcription Factors / metabolism
  • GATA5 Transcription Factor / genetics
  • GATA5 Transcription Factor / metabolism
  • Genome*
  • Molecular Sequence Data
  • Mutation
  • RNA Editing*
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism
  • Reverse Genetics / methods
  • Ribonucleases / genetics*
  • Ribonucleases / metabolism
  • Zebrafish / genetics*
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / metabolism

Substances

  • Etv2 protein, zebrafish
  • Fish Proteins
  • GATA Transcription Factors
  • GATA5 Transcription Factor
  • Gata4 protein, zebrafish
  • RNA, Guide
  • Zebrafish Proteins
  • gata5 protein, zebrafish
  • Ribonucleases