GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice

• Published in: Scientific reports Vol. 5; no. 1; p. 11406
• Main Authors: Takahashi, Gou, Gurumurthy, Channabasavaiah B, Wada, Kenta, Miura, Hiromi, Sato, Masahiro, Ohtsuka, Masato
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 22-06-2015; Nature Publishing Group
• Subjects: 42; 42/41; 631/1647/1511; 631/1647/2300/1850; 631/61/17/1511; 64; Animals; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Cas Systems - genetics; Electroporation; Electroporation - methods; Elongation Factor 2 Kinase - genetics; Embryo, Mammalian - cytology; Embryos; Female; Gene Targeting - methods; Genetic Engineering - methods; Genetically engineered organisms; Genetically modified organisms; Genome editing; Genomes; Green Fluorescent Proteins - genetics; Humanities and Social Sciences; Hypoxanthine Phosphoribosyltransferase - genetics; Mice; Mice, Inbred C57BL; Mice, Knockout; Microinjection; Microinjections - methods; mRNA; multidisciplinary; Nucleic acids; Oviduct; Oviducts - cytology; Pregnancy; RNA, Messenger - genetics; Science; Transgenes; Zygotes
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep11406

Microinjection is considered the gold standard technique for delivery of nucleic acids (NAs; transgenes or genome editing tools such as CRISPR/Cas9 systems) into embryos, for creating genetically modified organisms. It requires sophisticated equipment as wel as well-trained and highly skilled personnel to perform the micro-injection technique. Here, we describe a novel and simple microinjection-independent technique, called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). Using GONAD, we show that NAs (e.g., eGFP mRNA or Cas9 mRNA/sgRNAs) can be effectively delivered to pre-implantation embryos within the intact mouse oviduct by a simple electroporation method and result in the desired genetic modification in the embryos. Thus GONAD can bypass many complex steps in transgenic technology such as isolation of zygotes, microinjection of NAs into them and their subsequent transfer to pseudo-pregnant animals. Furthermore, this method can potentially be used for genome editing in species other than mice.