Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease.
Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability.
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA.
G Parker
GP
Flowers
Andrew T
AT
Timberlake
Kaitlin C
KC
McLean
James R
JR
Monaghan
Craig M
CM
Crews
D000558:Ambystoma mexicanum / Q000196:embryology / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054643:INDEL Mutation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D012038:Regeneration / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D058727:RNA, Small Untranslated
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases; D058727:RNA, Small Untranslated
Axolotl; CRISPR; Mutagenesis; Regeneration