title: Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.
abstract: Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan. masasato@ms.kagoshima-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan. k3326891@kadai.jp.
authors.forename: Miyu
authors.initials: M
authors.lastname: Koriyama
authors.affiliation: Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan. kettle@affrc.go.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Department of Cardiovascular Research, Graduate school of Medicine, Shinshu University, Nagano 390-8621, Japan. tsakurai@shinshu-u.ac.jp.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan. inada@dent.kagoshima-u.ac.jp.
authors.forename: Emi
authors.initials: E
authors.lastname: Inada
authors.affiliation: Division of Pediatric Dentistry, Department of Oral Health Sciences, Course for Oral Life Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan. isaito@dent.niigata-u.ac.jp.
authors.forename: Issei
authors.initials: I
authors.lastname: Saitoh
authors.affiliation: Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan. snaka@ndmc.ac.jp.
authors.forename: Shingo
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan. kmiyoshi@agri.kagoshima-u.ac.jp.
authors.forename: Kazuchika
authors.initials: K
authors.lastname: Miyoshi
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods; D020868:Gene Silencing; D006801:Humans; D054643:INDEL Mutation; D009030:Mosaicism; D059645:Mutation Rate; D009865:Oocytes / Q000378:metabolism*; D010312:Parthenogenesis; D012333:RNA, Messenger / Q000235:genetics*; D013552:Swine
chemical_list: D012333:RNA, Messenger; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine
keywords: CRISPR/Cas9; biallelic KO; indel mutations; isolectin BS-I-B4; mRNA; microinjection; mosaicism; α-1,3-galactosyltransferase; α-Gal epitope