title: Generation of α-1,3-Galactosyltransferase-Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9-Mediated Knock-in of a Small Mutated Sequence and a Targeted Toxin-Based Selection System.
abstract: The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α-gal epitope synthesized by α-1,3-galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig-to-human xenotransplantation. We here employed the CRISPR/Cas9 system-mediated knock-in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800-bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α-gal epitope expression, we successfully obtained several knock-in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9-mediated HR to knock-in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Sato
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, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Kagoshima
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, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Saitoh
authors.affiliation: Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Inada
authors.affiliation: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Miyoshi
authors.affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, Nagano, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology*; D005700:Galactosyltransferases / Q000172:deficiency* / Q000235:genetics; D055879:Gene Knock-In Techniques / Q000662:veterinary*; D059765:Homologous Recombination / Q000235:genetics; D009154:Mutation / Q000235:genetics; D034421:Sus scrofa / Q000196:embryology*; D014162:Transfection / Q000662:veterinary
chemical_list: D000939:Epitopes; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine