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

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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 muta-genesis 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.

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Flowers, G. P., & Crews, C. M. (2015). Generating and identifying axolotls with targeted mutations using Cas9 RNA-guided nuclease. Methods in Molecular Biology, 1290, 279–295. https://doi.org/10.1007/978-1-4939-2495-0_22

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