Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 16, Issue 8, Pages 17838-17856Publisher
MDPI
DOI: 10.3390/ijms160817838
Keywords
alpha-Gal epitope; alpha-1,3-galactosyltransferase; CRISPR/Cas9; microinjection; mRNA; mosaicism; biallelic KO; indel mutations; isolectin BS-I-B-4
Funding
- Ministry of Education, Science, Sports, and Culture, Japan [15K07695, 25450475]
- Grants-in-Aid for Scientific Research [25450475, 25850226, 26670153] Funding Source: KAKEN
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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 -1,3-galactosyltransferase that synthesizes the -Gal epitope using parthenogenetically activated porcine oocytes. The lack of -Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B-4 (IB4), which binds specifically to the -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.
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