title: Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.
abstract: 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.
authors.affiliation: Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China.
authors.forename: Hanchao
authors.initials: H
authors.lastname: Gao
authors.forename: Chengjiang
authors.initials: C
authors.lastname: Zhao
authors.forename: Xi
authors.initials: X
authors.lastname: Xiang
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zhao
authors.forename: Zesong
authors.initials: Z
authors.lastname: Li
authors.forename: Dengke
authors.initials: D
authors.lastname: Pan
authors.forename: Yifan
authors.initials: Y
authors.lastname: Dai
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Hara
authors.forename: David K C
authors.initials: DK
authors.lastname: Cooper
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Cai
authors.forename: Lisha
authors.initials: L
authors.lastname: Mou
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D000831:Animals, Newborn; D000906:Antibodies / Q000737:chemistry; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D054885:Cumulus Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics*; D055786:Gene Knockout Techniques; D005838:Genotype; D007074:Immunoglobulin G / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology; D006899:Mixed Function Oxygenases / Q000235:genetics*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D013552:Swine / Q000235:genetics*; D014183:Transplantation, Heterologous
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase