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 |
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| Authors: | M Sato A Kagoshima I Saitoh E Inada K Miyoshi M Ohtsuka S Nakamura T Sakurai S Watanabe |
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| Institution: | 1. Section 2. of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan;3. 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;4. Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan;5. Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan;6. Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan;7. Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama, Japan;8. Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, Nagano, Japan;9. Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan |
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| 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. |
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