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

Area of Science:

• Developmental Biology
• Regenerative Medicine
• Molecular Genetics

Background:

• Investigating gene function during axolotl regeneration is crucial for understanding limb regrowth.
• Traditional gene targeting methods are labor-intensive and complex.
• CRISPR/Cas9 RNA-guided nuclease technology presents a more accessible tool for genetic manipulation.

Purpose of the Study:

• To establish a streamlined reverse genetics approach using CRISPR/Cas9 for axolotl regeneration studies.
• To detail a cost-effective and efficient method for generating targeted mutations in axolotls.

Main Methods:

• Identification of targetable sequences within genes of interest.
• Design and synthesis of unique guide RNAs (gRNAs).
• Microinjection of gRNAs with Cas9-encoding mRNA into axolotl embryos.
• Selection of successfully injected animals.
• Development of a PCR-based screening method for identifying mutagenized individuals.

Main Results:

• Demonstrated the feasibility of using CRISPR/Cas9 for targeted gene mutagenesis in axolotls.
• Developed a less labor-intensive and more cost-effective gene targeting protocol compared to Zinc finger nucleases (ZFNs) or Transcription activator-like effector nucleases (TALENs).
• Established an efficient PCR-based method for screening mutations.

Conclusions:

• CRISPR/Cas9 provides a powerful and accessible tool for reverse genetics in axolotl regeneration research.
• The described methodology simplifies the process of gene function analysis in this model organism.
• This approach accelerates the study of genetic mechanisms underlying axolotl regeneration.