An Accessible Protocol for the Generation of CRISPR-Cas9 Knockouts Using INDELs in Zebrafish

Methods Mol Biol. 2019;1920:377-392. doi: 10.1007/978-1-4939-9009-2_23.

Abstract

The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.

Keywords: CRISPR-Cas9; Genome editing; INDELS; Targeted mutations; Zebrafish.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems*
  • Computational Biology / methods
  • Female
  • Gene Editing*
  • Gene Knockout Techniques*
  • Gene Targeting* / methods
  • INDEL Mutation*
  • Male
  • RNA, Guide / chemical synthesis
  • RNA, Guide / genetics
  • Zebrafish / genetics*

Substances

  • RNA, Guide