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.

Fig. 1.

CRISPR/Cas9 system targeting pig GGTA1 and CMAH loci. (A) Schematic of sgRNAs targeting GGTA1 and CMAH loci. The target sgRNAs were designed in the last exon of the GGTA1 locus and the first exon of the CMAH locus. sgRNA-target sequences are labeled in lowercase and the protospacer adjacent motif (PAM) is underlined. (B) Surveyor assay (T7E1) for Cas9-mediated cleavage at GGTA1 and CMAH loci in PK15 cells.

Fig. 2.

Selection of GGTA1 and CMAH gene bi-allelic DKO cell colonies. (A) Cell colonies were selected and subjected to two rounds of T7E1 Surveyor assays. The first round (left, PCR products of the candidate colonies were digested by T7E1) excluded heterozygotes, and the second round (right, PCR products of the candidate colonies and wild-type cells were mixed and then digested by T7E1) identified the candidate homozygotes. * indicates T7E1-digested fragments of the #8 cell colony at the GGTA1 and CMAH loci. (B) PCR amplicons of GGTA1 (left) and CMAH (right) loci of the #8 cell colony and wild-type cells. (C) Genotypes of the #8 cell colony at the GGTA1 and CMAH loci. PAM sequence is underlined.

Fig. 3.

Generation of GGTA1 and CMAH gene DKO piglets by HMC. (A) A schematic description of GGTA1/CMAH Wuzhishan PFFs used as nuclear donors and subjected to HMC resulting in the birth of GGTA1 and CMAH DKO piglets. (B) Photograph of three GGTA1/CMAH DKO piglets. (C) PCR products (above) and genotype analyzed by Sanger sequencing (below) of the 11 cloned piglets at the GGTA1 loci. (D) PCR products (above) and genotype analyzed by Sanger sequencing (below) of the 11 cloned piglets at the CMAH loci.

Fig. 4.

Immunofluorescence and flow cytometry analysis of Gal and Neu5Gc expression in GGTA1/CMAH DKO pigs. (A–B) Immunofluorescence of various tissues from wild-type or GGTA1/CMAH DKO pigs stained with anti-Neu5Gc antibody (A) and BS-IB4 lectin (B) (Magnification × 200; Neu5Gc, red; Gal, green; Nuclei, blue). (C–D) Flow cytometry analysis of AECs (C) and PBMCs (D) from the pigs indicated in A-B stained with anti-Neu5Gc antibody and BS-IB4 lectin. Human umbilical vein endothelial cells (HUVECs) and human PBMCs (HPBMCs) were negative controls, respectively. The green lines represent negative controls; the red lines represent the experimental groups.

Fig. 5.

Human antibody binding and antibody-mediated complement-dependent cytotoxicity of cells from GGTA1/CMAH DKO pigs. (A–B) Flow cytometry analysis of human IgM and IgG binding to RBCs (A) and PBMCs (B) from wild-type (n = 3), GGTA1-KO (n = 3), and GGTA1/CMAH DKO (n = 3) pigs. (C) Flow cytometry analysis of cytotoxicity of PBMCs from GGTA1-KO (n = 3) and GGTA1/CMAH DKO (n = 3) pigs incubated with different human serum concentrations. (*P < 0.05, ** P < 0.01, *** P < 0.001).