Applying a Knock-In Strategy to Create Reporter-Tagged Knockout Alleles in Axolotls

Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the "knocking out" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.