Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

Abstract

Background: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs.

Results: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets.

Conclusions: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.

Keywords: CRISPR/Cas9; Electroporation; GGTA1; In vitro fertilization; Pig.

Fig. 1

Confirmation of the gRNA gene-targeting efficiency. a: gRNA sequences targeting the GGTA1 gene and genomic structure of the GGTA1 locus. b: Blastocyst formation rates of the electroporated zygotes. For each treatment group, four replicates with 199–243 oocytes per treatment were analyzed. Values of means ± SEM are shown. c: Percentage of blastocysts carrying mutations in the GGTA1 target region after zygote electroporation with the Cas9 protein and each gRNA targeting GGTA1. The percentage of mutant blastocysts was defined as the ratio of mutant blastocysts to the total blastocysts. Percentages of mutant blastocysts was analyzed by chi-squared tests. ^a–dValues with different superscripts differ significantly (p < 0.05) and labels containing the same letter mean no significant difference. d: Genotypes of blastocysts determined by TIDE. WT, wild-type; Biallelic, biallelic mutant; Mosaic, mosaic mutant. Numbers above the bars indicate the total number of blastocysts examined

Fig. 2

Deep sequence analysis of the GGTA1 target region in delivered piglets. *Nucleotides in blue and red represent the target sequences and PAM sequences of each gRNA, respectively. Nucleotides in green and yellow represent inserted and modified sequences, respectively. **The frequency was defined as the ratio of the number of amplicons to the total read number. ***The mutation rate was defined as the ratio of the total number of mutant amplicons to the total read number. WT, wild-type; ♂, male; ♀, female

Fig. 3

Off-target analysis of the delivered piglets via deep sequencing. a: Genome sequences and positions of possible off-target sites. Nucleotides in blue and red represent the target sequences and the PAM sequences of gRNA5, respectively. Nucleotides in green represent mismatches with the gRNA5 sequence. b: Frequency of the WT sequence at possible off-target sites

Fig. 4

Immunohistochemical assessment of wild-type and GGTA1 biallelic mutant piglets. The heart, lung, liver, pancreas, and kidney tissues derived from wild-type (WT) and GGTA1 biallelic mutant piglets (#1) were immunohistochemically stained for αGal (green) and counterstained with DAPI (blue). The scale bar in each panel represents 50 μm

Fig. 5

Genotype of major organs derived from piglets #1 analyzed by deep sequencing. Frequency of introduced mutations in selected organs

Fig. 6

Comparison of the expression of αGal epitope in GGTA1 mutant piglets with various genotypes by immunohistochemical assessment. The ear biopsy derived from wild-type (WT) and GGTA1 biallelic mutant piglets (#2, #3, #4, and #5) were immunohistochemically stained for αGal (green) and counterstained with DAPI (blue). The scale bar in each panel represents 50 μm

Fig. 7

Analysis of the genome sequences of F1 piglets. The alignment of sequences from F1 piglets is shown. The nucleotides in blue and red represent the target sequences and the PAM sequences of gRNA5, respectively. The nucleotides in green represent the inserted sequences and those in yellow represent the modified sequences