Generating and identifying axolotls with targeted mutations using Cas9 RNA-guided nuclease.
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.
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 06511, USA.
G Parker
GP
Flowers
Craig M
CM
Crews
D000558:Ambystoma mexicanum / Q000196:embryology / Q000235:genetics*; D000818:Animals; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D003851:Deoxyribonucleases / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics*; D005260:Female; D008297:Male; D008845:Microinjections; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D004247:DNA; D003851:Deoxyribonucleases; D004720:Endonucleases