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 pigto-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.

Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos

“…These oocytes have a low surface-to-volume ratio, making it difficult for LF2000 to move across the cell plasma membranes, resulting in low toxicity [ 24 , 25 ]. However, all mutated blastocysts derived from ZP-free oocytes treated with LF2000 were genetically mosaic upon the use of gRNAs targeting either PDX1 or GGTA1 , whose gene editing efficiency was confirmed in our previous study [ 19 , 20 ]. These results indicate that the timing of RNP introduction into oocytes may not be proper for producing biallelic mutant blastocysts.…”

“…In our lipofection-mediated system, gene-editing efficiency was insufficient compared with electroporation and microinjection-mediated gene editing [ 20 , 37 ], resulting in a low number of biallelic mutant blastocysts. Our previous study demonstrated that the results of electroporation- and microinjection-mediated gene editing were also affected by the concentration of CRISPR/Cas9 components, voltage strength during electroporation, and the developmental stage of zygotes/embryos [ 37 , 38 , 39 ].…”

“…gRNAs targeting PDX1 (5′-TGGCGAGGAGCAGTACTACG-3′), which have been confirmed to show high genome editing efficiency [ 19 ], and gRNA targeting GGTA1 (5′- TGTTTTGGGAATACATCAAC-3′), which was used to produce GGTA1 -deficient pigs [ 20 ], were used to evaluate the gene editing of porcine oocytes by lipofection during IVF. Matured oocytes were denuded from the cumulus cells by mechanical pipetting with 0.1% ( w / v ) hyaluronidase; subsequently, the ZP of matured oocytes was removed prior to IVF.…”

Improvements in Gene Editing Technology Boost Its Applications in Livestock

“…More recently, farm animals with point mutations and gene insertions (KI) have been successfully produced using ssODN donor sequences, CRISPR/Cas9 base editing and CRISPR/Cas9 nickase approaches ( Table 3 ). The applications of gene editing technologies for generation of livestock are very diverse, ranging from enhancing important production traits such as meat, milk, and fiber production ( Deng et al, 2014 ; Crispo et al, 2015 ; Hu et al, 2017 ; Zhou et al, 2017 ; Zhang R. et al, 2020 ) to improving disease resistance, health, reproductive efficiency, facilitating animal welfare, and developing new biomedical models to better understand the etiology of diseases and develop novel mechanism-based therapeutic approaches ( Vilarino et al, 2017 ; Fan et al, 2018a , b ; Tu et al, 2019 ; Tanihara et al, 2020 ).…”

Methodologies and Challenges for CRISPR/Cas9 Mediated Genome Editing of the Mammalian Brain

“…These improvements hold great promise for a rapid generation of transgenic mouse models, which will allow behavioral and functional screening at the level of F0 mice, and thus circumvent the need for long periods of cross-breeding. The genome editing of fertilized eggs by electroporation has been performed not only in mice but also in rats (Kaneko, 2017) and pigs (Tanihara et al, 2016(Tanihara et al, , 2020. In particular, it has been reported that the electroporation method for editing the genome of porcine fertilized eggs has several advantages over the microinjection method, such as shorter production time and reduced pre-and postnatal death rates (Tanihara et al, 2016).…”