Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning

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  • GAO Hanchao
    Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
  • ZHAO Chengjiang
    Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
  • XIANG Xi
    BGI Ark Biotechnology (BAB), Shenzhen 518083, China
  • LI Yong
    BGI Ark Biotechnology (BAB), Shenzhen 518083, China
  • ZHAO Yanli
    Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China
  • LI Zesong
    Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China
  • PAN Dengke
    Key Laboratory of Farm Animal Genetic Resource and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
  • DAI Yifan
    Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China
  • HARA Hidetaka
    Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL 35233, USA
  • COOPER David K.C.
    Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL 35233, USA
  • CAI Zhiming
    Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China
  • MOU Lisha
    Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China

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<p> Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.</p>

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