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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.

international journal of molecular sciences

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations