title: The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the α-1,3-galactosyltransferase gene in porcine embryonic fibroblasts.
abstract: BACKGROUND
  The recent development of the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled genome editing of mammalian genomes including those of mice and human; however, its applicability and efficiency in the pig have not been studied in depth. Here, using the CRISPR/Cas9 system, we aimed to destroy the function of the porcine α-1,3-galactosyltransferase (α-GalT) gene (GGTA1) whose product is responsible for the synthesis of the α-Gal epitope, a causative agent for hyperacute rejection upon pig-to-human xenotransplantation.
  
  
  METHODS
  Porcine embryonic fibroblasts were transfected with a Cas9 expression vector and guide RNA specifically designed to target GGTA1. At 4 days after transfection, the cells were incubated with IB4 conjugated with saporin (IB4SAP), which eliminates α-Gal epitope-expressing cells. Therefore, the cells surviving after IB4SAP treatment would be those negative for α-Gal epitope expression, which in turn indicates the generation of GGTA1 biallelic knockout (KO) cells.
  
  
  RESULTS
  Of the 1.0 × 10(6) cells transfected, 10-33 colonies survived after IB4SAP treatment, and almost all colonies (approximately 90%) were negative for staining with red fluorescence-labeled IB4. Sequencing of the mutated portion of GGTA1 revealed a frameshift of the α-GalT protein. Porcine blastocysts derived from the somatic cell nuclear transfer of these α-Gal epitope-negative cells also lacked the α-Gal epitope on their surface.
  
  
  CONCLUSIONS
  These results demonstrated that the CRISPR/Cas9 system can efficiently induce the biallelic conversion of GGTA1 in the resulting somatic cells and is thus a promising tool for the creation of KO cloned piglets.
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.forename: Kazuchika
authors.initials: K
authors.lastname: Miyoshi
authors.forename: Yozo
authors.initials: Y
authors.lastname: Nagao
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nishi
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.forename: Shingo
authors.initials: S
authors.lastname: Nakamura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sakurai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D005347:Fibroblasts; D005700:Galactosyltransferases / Q000235:genetics*; D055786:Gene Knockout Techniques; D005819:Genetic Markers; D008297:Male; D013552:Swine / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine
keywords: 3-galactosyltransferase; CRISPR/Cas9; biallelic knockout; porcine embryonic fibroblasts; somatic cell nuclear transfer; targeted toxin; α-1