Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

• DOI: 10.1242/dev.105072
• Publication Date: 2014-04-25T02:05:08Z
• Authors: G. Parker Flowers; Andrew T. Timberlake; Kaitlin C. Mclean; James R. Monaghan; Craig M. Crews
• Keywords: Ambystoma mexicanum; Brachyury

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs adults. The mystery of why but not other animals, possess this ability has for generations captivated scientists seeking to induce phenomenon in vertebrates. Although many recent advances molecular biology have allowed limb regeneration and tissue repair be investigated increasing detail, toolkit study process been limited. Here, we report that CRISPR-Cas9 RNA-guided nuclease system efficiently create mutations at targeted sites within genome. We identify individual animals treated with nucleases mutation frequencies close 100% sites. employ technique functionally ablate EGFP expression transgenic recapitulate developmental phenotypes produced by loss conserved gene brachyury. Thus, advance allows a reverse genetic approach will undoubtedly provide invaluable insight into mechanisms salamanders' unique regenerative ability.