earticle

영어

The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Specific gene knock out and knock in system in pig holds a bright future for the study of complex disease. Using CRISPR/Cas9 system we targeted OCT4gene specifically knock-out the OCT-4 gene during a preimplantation stage. We injected both Cas9 RNA and sgRNA (OCT-4 targeted in exon2) for deletion of specific site, and also injected Cas9 RNA and two sgRNAs (OCT-4 targeted in exon2 and exon5) for deletion of large scale site in OCT-4 sequence. Injection of cas9 and sgRNA against Oct4 decrease percentages of Oct4-positive embryo to 37～50% of total embryo, while ~100% of control embryo show clear Oct4 immunostaining We checked the presence of mutation near guide sequence using PCR and DNA sequencing, significant portion of clones (20% in Exon 2 and 50% in Exon 5) has insertion/deletion near protospacer adjacent motif(PAM). Different target sites have several frequency of deletion but, different concentration of sgRNA was no difference. Oct4 mRNA levels is dramatically decreased at 8 cell stages and barely detectable in BL stages, while other genes including Sox2, Nanog and CDX2 mRNA levels did not affected. Also, the combination of two sgRNAs can be large scale deletion(about 1.8kb) in the same chromosome. In conclusion, CRISPR/CAS9 system provides a good tool for gene function study by deleting target gene in pig.