Abstract
Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.
Highlights
Gene modification using homologous recombination (HR)-based gene targeting is a powerful technique for exploring gene function and producing human disease models
These were microinjected into the cytoplasm of parthenogenetically activated (PA) porcine oocytes to examine the mode of action of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene disruption in porcine parthenotes in terms of loss of α-Gal epitope expression
In which the embryo 2m++ shown in Figure 2A is further magnified, the green fluorescent area enclosed by a dotted line showed weaker AF594-IB4 staining, indicating a correlation between the green fluorescent signal and reduced α-Gal epitope expression. These findings suggest that several porcine blastocysts derived from CRISPR/Cas9 mRNA-injected oocytes are comprised of at least three types of cells, biallelic KO, monoallelic KO, and wild-type cells
Summary
Gene modification using homologous recombination (HR)-based gene targeting is a powerful technique for exploring gene function and producing human disease models. We prepared guide RNA specific to exon 4 (containing the ATG site) of GGTA1, which has been proven to be effective during CRISPR/Cas9-meditated gene disruption [27], together with mRNAs for enhanced green fluorescent protein (EGFP; used for monitoring the fate of cytoplasmically injected mRNA) and human Cas. We prepared guide RNA specific to exon 4 (containing the ATG site) of GGTA1, which has been proven to be effective during CRISPR/Cas9-meditated gene disruption [27], together with mRNAs for enhanced green fluorescent protein (EGFP; used for monitoring the fate of cytoplasmically injected mRNA) and human Cas9 These were microinjected into the cytoplasm of parthenogenetically activated (PA) porcine oocytes to examine the mode of action of CRISPR/Cas9-mediated gene disruption in porcine parthenotes in terms of loss of α-Gal epitope expression. We employed whole genome amplification (WGA) which potentially allows multiple PCR-based analyses from the small amount of genomic DNA isolated from a single blastocyst [39,42]
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