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

摘要: Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA CRISPR/Cas9-related components. Given the relatively long gestation periods and high cost housing, establishment a single blastocyst-based assay for rapid optimization above system is required. As proof-of-concept, we attempted to disrupt gene (GGTA1) encoding α-1,3-galactosyltransferase that synthesizes α-Gal epitope using parthenogenetically activated porcine oocytes. The lack expression can be monitored staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically epitope. When oocytes were injected guide RNA specific GGTA1 together enhanced green fluorescent protein (EGFP) human Cas9 mRNAs, 65% (24/37) developing blastocysts exhibited fluorescence, although almost all (96%, 23/24) showed mosaic pattern. Staining IB4 revealed area often had reduced binding activity IB4. Of 16 samples tested, six (five one non-fluorescent blastocysts) indel mutations, suggesting correlation between EGFP mutation induction. Furthermore, it suggested mRNAs might lead production piglets cells harboring various types.