The reconstructed oocytes were parthenogenetically activated by incubation in 7.5 mol/L ionomycin (I3909, Sigma, St Louis, MO, USA) for 10 min followed by incubation in 2 mmol/L 6-dimethylamino purine (6-DMAP; d2629, Sigma, St Louis, MO, USA) for 4 h. Activated oocytes with 1 PN were injected G1 gRNA, Cas9 mRNA, and the ssDNAoligo into the cytoplasm 5–6 h after activation. The survived reconstructed embryos were cultured in microdrops of G-1 medium (Vitrolife, VitrolifeSweeden AB Göteborg, Sweeden) at 37°C in a humidified atmosphere of 6% CO2, 5% O2, and 89% N2 for 42 h. Blastomere of reconstructed embryos were individually aspirated out of ZP by the pipette.