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Development of PCR based approach to detect potential mosaicism in porcine embryos

Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, 24061, USA College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea Division of Animal Science, University of Missouri, Columbia, MO, 65211, USA Division of Reproductive & Developmental Sciences, Oregon Health & Science University, Beaverton, OR, 97006, USA Original Article J Anim Reprod Biotechnol 2020;35:323-328 pISSN: 2671-4639 • eISSN: 2671-4663 https://doi.org/10.12750/JARB.35.4.323 JARB Journal of Animal Reproduction and Biotechnology

. During the last 20 years, genetically engineered (GE) pigs have been mainly produced through manipulating genes in the somatic cells, followed by somatic cell nuclear transfer (SCNT) (Dai et al., 2002;Lai et al., 2002;Choi et al., 2017;Jeon and Rho, 2020). Although successful, GE pigs produced via SCNT often present health complications Piedrahita et al., 2004;Lee et al., 2005). Alternatively, direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos can introduce targeted modifications.
GE pigs produced via direct injection of CRISPR/Cas9 system do not possess health complications at birth; however, the pigs may carry more than two alleles if targeted modifications occur after cell division. The mosaicism is considered to be a shortcoming of the approach because the phenotype of mosaic pigs often does not represent targeted modifications and the genotype of progeny derived from the mosaic pigs is difficult to predict.
The frequency of mosaicism is difficult to assess because capturing all alleles from developing embryos is extremely challenging. Establishing an effective system to detect the frequency of mosaicism will assist us to design CRISPR/Cas9 systems with minimal mosaicism. Typically, genomic PCR followed by sequencing is performed to detect potential mosaicism using blastocysts . It is easier to conduct the PCR analysis using blastocysts because sufficient amount of genetic content can be obtained. However, PCR amplification of genomic DNA from blastocysts will not be able to calculate the degree of mosaicism and detect all possible alleles as PCR amplifications can be biased from a pool of genomic DNA. Single cell sequencing approach may be used to detect the level of mosaicism in blastocysts; however, cost and labor associated with the analysis are extensive.
In this study, we attempted to develop a simplified method to identify genomic information of 4-cell stage embryos by genotyping single blastomeres in the embryos. Specifically, we amplified and sequenced genomic region of TET1 gene from single blastomeres of 4-cell stage embryos to verify the feasibility of detecting mosaicism in early stage embryos. Our procedure will greatly benefit efforts to prevent mosaicism when engineering the genome using CRISRP/Cas9.

MATERIALS AND METHODS
Experiments of this study including in vitro embryo production and genotyping, were conducted based on our previous reports (Lei et al., 2016;Ryu and Lee, 2017;Uh et al., 2020). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.

In vitro maturation of pig oocytes
Sow ovaries obtained from a local abattoir were transported to the laboratory and washed with DPBS. Oocytes were aspirated from the ovaries using an 18-guage needle attached to syringes. Aspirates were washed with TL-Hepes medium three times and placed in petri dishes.
Fresh semen obtained from boars were mixed with semen extender and stored at 17℃ up to one week. After placing the oocytes in fertilization medium, 1 mL semen was diluted by adding 9 mL DPBS supplemented with 0.1% BSA, and then washed at 720 × g for 3 min by centrifugation.
After 3 washing steps, the sperm pellet was resuspended with fertilization medium, and 50 µL of the resuspension was added to the droplet containing oocytes. The final concentration of sperm in the droplet was approximately 0.25 × 10 6 /mL. Oocytes and sperms were coincubated in the fertilization medium at 38.5℃, 5% CO 2 for 5 h, then moved to culture droplets containing PZM3 medium (Yoshioka et al., 2002) and incubated at 38.5℃, 5% CO 2 , 5% O 2 .

Successful PCR amplification of a target gene from a single blastomere
We examined whether PCR amplification from a small amount gDNA is feasible. Similar to our previous study (Uh et al., 2020), a region of TET1 gene was successfully amplified from a single blastocyst (Fig. 2). Sensitivity of the PCR amplification was high enough to amplify the TET1 region from a single 4-cell stage embryo, i.e. four copies of genomic DNA. To examine the feasibility of amplifying the target region from a single blastomere, 4 blastomeres were separated from a 4-cell stage embryo. Genomic DNAs derived from the individual blastomeres were used for PCR, and target region was successfully amplified from the blastomeres. This result indicates that a single copy DNA in a single blastomere of a 4-cell stage embryo gDNA is sufficient to amplify a target genomic region through conventional PCR approach.

Genotyping of single blastomeres from a 4-cell stage embryo
The PCR amplicons obtained from the whole 4-cell   of TET1 gene were also observed in Sanger sequencing of two blastomeres of the 4-cell embryo ( Fig. 3C and 3D).
In sequencing of other two blastomeres, there were some baseline noise, but major peaks were completely matched to the target sequence ( Fig. 3E and 3F). This result suggests that mutation types generated by introducing CRIS-PR/Cas9 into zygotes can be determined in early stage embryos. The approach of this study could promote the efficiency of producing genetically engineered animals by detecting mosaicism in relatively early stage of preimplantation development.

DISCUSSION
Prevalence of mosaic genotype is one of the major disadvantages to utilize direct injection of CRISPR/Cas9 system into developing embryos because animals carrying mosaic genotypes often interfere with phenotyping and breeding the founder animals (Lei et al., 2016;. Developing an effective approach to detect the frequency of mosaicism assist us to design and optimize CRISPR/Cas9 systems that introduce mosaicism at minimal level. In this study, we successfully genotyped a single blastomere from porcine 4-cell stage embryos, demon-strating that it is possible to perform genomic PCR on a single copy of genomic DNA. We propose that PCR-based genotyping of single blastomere is an efficient approach to identify genomic-modifications in early stage embryos after direct injection of CRISPR/Cas9 system into presumable zygotes. Previous reports suggest that microinjection timing (Sato et al., 2018) and CRISPR/Cas9 concentration  can influence the frequency of mosaicism in CRISPR/Cas9 injected embryos. For example, microinjection of CRISPR/Cas9 into germinal vesicle-stage oocytes appears to produce non-mosaic transgenic embryos (monoallelic knockouts) (Su et al., 2019). Another study identified optimal microinjection conditions in terms of CRISPR/Cas9 concentration and type, injection time, and embryos (Liu et al., 2016). Rapidly detecting the frequency of mosaicism at a low cost can assist in developing condi-  Unlike whole genome sequencing approach, our PCRbased detection of mosaicism is simple and can deliver the frequency of mosaicism in just a few days. Although detecting mosaicism using whole genome sequencing offers an extensive snapshot of possible alleles in embryos or animals, the method requires library preparation and bioinformatic analysis of sequencing information, which can take months. Alternatively, our PCR-based approach does not require specific equipment or expertise to detect mosaicism and ability to identify mosaicism after CRISPR/ Cas9 injection can be comparable to the whole genome sequencing. Due to the lack of knockout embryos at the time of this study, no targeted event has been detected using the approach. However, our system should be instantly applied to embryos carrying targeted modifications.
In conclusion, we demonstrated that Sanger sequencing of PCR amplicons obtained from single blastomere DNA in 4-cell stage embryo is a feasible method for genotyping of mammalian embryos. Our technique opens the doors to future research that can determine optimal microinjection conditions for minimizing or eliminating mosaicism, therefore, improving the use of CRISPR/Cas9-mediated genome modification in mammalian embryos.

CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.