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CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

Background and Aim: The antioxidant effects of ergothioneine (EGT) on in vitro culture of porcine zygote are not well established. The study investigated the impact of EGT supplementation on meiotic competence and development of porcine oocytes., Materials and Methods: The impact of EGT concentrations (0, 5, 10, 25, 50, and 100 μM) during in vitro maturation (IVM) on the progression of meiotic maturation, fertilization, and blastocyst formation in porcine oocytes was assessed. The DNA fragmentation level was evaluated to determine oocyte and blastocyst quality., Results: The proportion of metaphase II oocytes was significantly greater (p < 0.05) in EGT-cultured oocytes than in control oocytes, regardless of the EGT concentration, and those oocytes with 10 μM or more EGT had fewer DNA-fragmented nuclei (p < 0.05). Blastocysts derived from oocytes cultured with 10 μM EGT had the highest proportion (p < 0.05), while those from control oocytes or oocytes cultured with 50 μM or less EGT had significantly higher proportions. Despite EGT supplementation, there were no noticeable differences in total cell numbers and DNA fragmentation indices in the derived blastocysts., Conclusion: Supplementing with EGT during IVM leads to better oocyte maturation, quality, and embryonic development due to decreased DNA fragmentation. The present study failed to elucidate the mechanism of DNA fragmentation reduction by EGT. More research needs to be conducted to explore the antioxidant mechanism of EGT., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Nagahara, et al.)