Generating and identifying axolotls with targeted mutations using Cas9 RNA-guided nuclease

Methods Mol Biol. 2015:1290:279-95. doi: 10.1007/978-1-4939-2495-0_22.

Abstract

The CRISPR/Cas9 RNA-guided nuclease now enables a reverse genetics approach to investigate the function of genes of interest during regeneration in the axolotl. The process of generating the constructs necessary for targeting a gene of interest is considerably less labor intensive than for other methods of targeted mutagenesis such as Zinc finger nucleases or Transcription activator-like effector nucleases. Here, we describe the identification of targetable sequences in the gene of interest, the construction of unique guide RNAs, the microinjection of these RNAs with Cas9-encoding mRNA, the selection of well-injected animals, and an inexpensive, PCR-based method for identifying highly mutagenized animals.

MeSH terms

  • Ambystoma mexicanum / embryology
  • Ambystoma mexicanum / genetics*
  • Animals
  • DNA / genetics
  • DNA / isolation & purification
  • Deoxyribonucleases / metabolism*
  • Embryo, Nonmammalian / metabolism
  • Endonucleases / genetics*
  • Female
  • Male
  • Microinjections
  • Mutagenesis, Site-Directed / methods*
  • Mutation*
  • Polymerase Chain Reaction
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Messenger
  • DNA
  • Deoxyribonucleases
  • Endonucleases