Chapters authored
Recent Advance in Genome Editing-Based Gene Modification in Pigs
Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, in vitro electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transfer-derived embryos that have been reconstituted with normal nuclei.
Part of the book: Reproductive Biology and Technology in Animals
Recent Advances in In Vivo Genome Editing Targeting Mammalian Preimplantation Embryos
CRISPR-based genome engineering has been widely used for producing gene-modified animals such as mice and rats, to explore the function of a gene of interest and to create disease models. However, it always requires the ex vivo handling of preimplantation embryos, as exemplified by the microinjection of genome editing components into zygotes or in vitro electroporation of zygotes in the presence of genome editing components, and subsequent cultivation of the treated embryos prior to egg transfer to the recipient females. To avoid this ex vivo process, we have developed a novel method called genome-editing via oviductal nucleic acids delivery (GONAD) or improved GONAD (i-GONAD), which enables in situ genome editing of zygotes present in the oviductal lumen of a pregnant female. This technology does not require any ex vivo handling of preimplantation embryos or preparation of recipient females and vasectomized males, all of which are often laborious and time-consuming. In this chapter, recent advances in the development of GONAD/i-GONAD will be described.
Part of the book: CRISPR Technology