Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

Although trisomy 16 is commonly detected in spontaneous abortions and accounts for over 30% of cases of autosomal trisomy detected after spontaneous abortion, trisomy 16 mosaicism is rarely detected by amniocentesis in the second trimester. Here, we report a case of level III trisomy 16 mosaicism (47,XX,+16[8]/46,XX[31]) diagnosed by cytogenetic analysis of independently cultured amniotic fluid cells. The female baby was delivered at full term with low birth weight and intrauterine growth retardation, and interestingly, her karyotype was normal (46,XX). Given the difficulty in predicting the outcomes of fetuses with this mosaicism, it is recommended to inform the possibility of mosaicisms including this trisomy 16 mosaicism during prenatal genetic diagnosis and genetic counseling for parents.