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

International Journal of Molecular Sciences

Masahiro Sato⁴  Miyu Koriyama⁸  Satoshi Watanabe³  Masato Ohtsuka⁶  Takayuki Sakurai¹  Emi Inada⁵  Issei Saitoh⁷  Shingo Nakamura²  Kazuchika Miyoshi⁸ 

[1] Department of Cardiovascular Research, Graduate school of Medicine, Shinshu University, Nagano 390-8621, Japan; E-Mail:;Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan; E-Mail:;Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan; E-Mail:;Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan;Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan; E-Mail:;Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan; E-Mail:;Division of Pediatric Dentistry, Department of Oral Health Sciences, Course for Oral Life Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan; E-Mail:;Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan; E-Mails:

关键词: α-Gal epitope;    α-1;    3-galactosyltransferase;    CRISPR/Cas9;    microinjection;    mRNA;    mosaicism;    biallelic KO;    indel mutations;    isolectin BS-I-B₄;   

DOI  :  10.3390/ijms160817838

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.