Cas9-based genome editing in zebrafish

Methods Enzymol. 2014;546:377-413. doi: 10.1016/B978-0-12-801185-0.00018-0.

Abstract

Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unprecedented efficacy and facility in a wide variety of biological systems. In zebrafish, specifically, studies have shown that Cas9 can be directed to user-defined genomic target sites via synthetic guide RNAs, enabling random or homology-directed sequence alterations, long-range chromosomal deletions, simultaneous disruption of multiple genes, and targeted integration of several kilobases of DNA. Altogether, these methods are opening new doors for the engineering of knock-outs, conditional alleles, tagged proteins, reporter lines, and disease models. In addition, the ease and high efficiency of generating Cas9-mediated gene knock-outs provides great promise for high-throughput functional genomics studies in zebrafish. In this chapter, we briefly review the origin of CRISPR/Cas technology and discuss current Cas9-based genome-editing applications in zebrafish with particular emphasis on their designs and implementations.

Keywords: CRISPR; Cas9; Chromosomal conversion; Chromosomal deletion; Gene-editing; Genome engineering; Homology-directed repair; Knock-in; Knock-out; Targeted integration; Targeted mutagenesis; Zebrafish.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Gene Targeting / methods*
  • Genetic Engineering / methods
  • Genome
  • INDEL Mutation
  • RNA, Guide / genetics
  • Zebrafish / genetics*

Substances

  • RNA, Guide