title: GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos.
abstract: Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD (Genome editing via Oviductal Nucleic Acids Delivery) that does not require all the three critical steps of transgenesis and therefore relieves the bottlenecks of widely used animal transgenic technologies. Here we provide protocols for the GONAD system.
authors.affiliation: Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska.|These authors contributed equally to this work.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.|Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Hokkaido, Japan.|These authors contributed equally to this work.
authors.forename: Gou
authors.initials: G
authors.lastname: Takahashi
authors.affiliation: Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Hokkaido, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Wada
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miura
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D018274:Electroporation / Q000379:methods; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008845:Microinjections; D010057:Oviducts / Q000378:metabolism*; D015203:Reproducibility of Results; D015053:Zygote / Q000378:metabolism
keywords: CRISPR/Cas9; GONAD; gene delivery; genome editing; in vivo electroporation; microinjection; transgenic