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Deprecated: Implicit conversion from float 298.79999999999995 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Int+J+Mol+Sci 2015 ; 16 (8): 17838-56 Nephropedia Template TP
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Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations #MMPMID26247938
Sato M; Koriyama M; Watanabe S; Ohtsuka M; Sakurai T; Inada E; Saitoh I; Nakamura S; Miyoshi K
Int J Mol Sci 2015[Aug]; 16 (8): 17838-56 PMID26247938show ga
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-B4 (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.