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

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

Author information

1
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 06511, USA.

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.

PMID:
25740494
DOI:
10.1007/978-1-4939-2495-0_22
[Indexed for MEDLINE]

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