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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
Author(s) -
Sato Masahiro,
Miyoshi Kazuchika,
Nagao Yozo,
Nishi Yohei,
Ohtsuka Masato,
Nakamura Shingo,
Sakurai Takayuki,
Watanabe Satoshi
Publication year - 2014
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/xen.12089
Subject(s) - crispr , epitope , biology , cas9 , transfection , microbiology and biotechnology , genome editing , gene targeting , gene knockout , hek 293 cells , electroporation , xenotransplantation , gene , transplantation , genetics , antibody , medicine , surgery
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 ( GGTA 1 ) 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 GGTA 1 . At 4 days after transfection, the cells were incubated with IB 4 conjugated with saporin ( IB 4 SAP ), which eliminates α‐Gal epitope‐expressing cells. Therefore, the cells surviving after IB 4 SAP treatment would be those negative for α‐Gal epitope expression, which in turn indicates the generation of GGTA 1 biallelic knockout ( KO ) cells. Results Of the 1.0 × 10 6 cells transfected, 10–33 colonies survived after IB 4 SAP treatment, and almost all colonies (approximately 90%) were negative for staining with red fluorescence‐labeled IB 4. Sequencing of the mutated portion of GGTA 1 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 GGTA 1 in the resulting somatic cells and is thus a promising tool for the creation of KO cloned piglets.