谷原 史倫

BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

BACKGROUND: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation. OBJECTIVE: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes. MATERIALS AND METHODS: In vitro-derived bovine zygotes were electroporated with the CRISPR/Cas9 system immediately (Vitrified-EP) or 2 h after incubation (Vitrified-2h-EP) following vitrification and warming, or electroporated before vitrification (EP-vitrified). RESULTS: The development rates of vitrified-warmed zygotes were significantly lower (p < 0.05) than those of control zygotes that were not vitrified. Moreover, no differences were observed in the mutation rates and mutation efficiency of the blastocysts resulting from electroporated zygotes, irrespective of the timing of electroporation treatment. CONCLUSION: Our results suggest that vitrification before and after electroporation treatments does not affect the genome editing of zygotes.

Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine insulin (INS) gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation. However, electroporation has not been used to generate point-mutated pigs yet. In the present study, we introduced a point mutation into porcine zygotes via electroporation using the CRISPR/Cas9 system to generate INS point-mutated pigs as suitable islet donors. We first optimized the efficiency of introducing point mutations by evaluating the effect of Scr7 and the homology arm length of ssODN on improving homology-directed repair-mediated gene modification. Subsequently, we prepared electroporated zygotes under optimized conditions and transferred them to recipient gilts. Two recipients became pregnant and delivered five piglets. Three of the five piglets carried only the biallelic frame-shift mutation in the INS gene, whereas the other two successfully carried the desired point mutation. One of the two pigs mated with a WT boar, and this desired point mutation was successfully inherited in the next F1 generation. In conclusion, we successfully established genetically engineered pigs with the desired point mutation via electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.

Pigs are excellent large animal models owing to their several physiological and anatomical similarities to humans. Somatic cell nuclear transfer using gene-modified cells is the mainstream approach for generating genetically modified pigs. Recent advances in improving gene editors such as the CRISPR/Cas9 system have enabled direct gene modification in zygotes/embryos. Here, we describe the gene editing by electroporation of Cas9 protein (GEEP) method, an optimized electroporation-mediated method for the introduction of CRISPR/Cas9 into porcine zygotes/embryos. The simplicity and micromanipulation-free procedures are the major advantages of this method.

Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.