Generating and Identifying Axolotls with Targeted Mutations Using Cas9 RNA-Guided Nuclease.
Salamanders in Regeneration Research
Methods in molecular biology, January 2015
G Parker Flowers, Craig M Crews, G. Parker Flowers, Craig M. Crews
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.
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