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.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONFLICTS OF INTEREST
ACKNOWLEDGEMENTS
AUTHOR CONTRIBUTIONS
AUTHOR’S POSITION AND ORCID NO
REFERENCES

• Jongki Cho(Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, 24061, USA, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Kyungjun Uh(Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, 24061, USA, Division of Animal Science, University of Missouri, Columbia, MO, 65211, USA)
• Junghyun Ryu(Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, 24061, USA, Division of Reproductive & Developmental Sciences, Oregon Health & Science University, Beaverton, OR, 97006, USA)
• Xun Fang(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Seonggyu Bang(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Kiho Lee(Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, 24061, USA, Division of Animal Science, University of Missouri, Columbia, MO, 65211, USA) Corresponding author