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Salamanders in Regeneration Research

Volume 1290 of the series Methods in Molecular Biology pp 279-295

Date:

Generating and Identifying Axolotls with Targeted Mutations Using Cas9 RNA-Guided Nuclease

  • G. Parker FlowersAffiliated withDepartment of Molecular, Cellular and Developmental Biology, Yale University
  • , Craig M. CrewsAffiliated withDepartment of Molecular, Cellular and Developmental Biology, Yale University Email author 

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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.

Key words

Transgenesis Salamander CRISPR Limb regeneration Guide RNA