i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

The recent development of genome editing technologies has enabled the creation of genome‐edited animals, with alterations at the desired target locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for this purpose because it is simpler and more efficient than other genome editing technologies. The conventional methods for creation of genome‐edited animals involve ex vivo handling of embryos (zygotes) for microinjection or in vitro electroporation. However, this process is laborious and time‐consuming, and relatively large numbers of animals are used. Furthermore, these methods require specialized skills for handling embryos. In 2015, we reported a novel method for the creation of genome‐edited animals without ex vivo handling of embryos. The technology known as Genome‐editing via Oviductal Nucleic Acids Delivery (GONAD) involved intraoviductal instillation of genome editing components into a pregnant female and subsequent in vivo electroporation of an entire oviduct. The genome editing components present in the oviductal lumen are transferred to preimplantation embryos in situ for introducing insertion or deletion (indel) mutations at the desired loci. This technology was further improved by optimizing several parameters to develop improved GONAD (i‐GONAD) for the efficient generation of mutant or knock‐in animals. In this review, we discuss the historical background, potential applications, advantages, and future challenges of GONAD/i‐GONAD technology. i‐GONAD method enables genome editing of embryos without taking them out from the female reproductive system. Because this method does not require skilled personnel or sophisticated equipment, its utility is not restricted to specialized core laboratories and can be used for generating animal models in any laboratories.