Genome Editing of Frozen Embryos

Genome editing of frozen-warmed embryos was performed using the TAKE method^{18 (link)–20 (link)}. A super-electroporator NEPA21 (NEPA GENE Co. Ltd., Chiba, Japan) was used to introduce nucleases. The nuclease solution comprised 100 μg/mL of Cas9 protein (Integrated DNA Technologies Inc. Coralville, IA, USA) and 500 μg/mL of dual RNA (mixture of crRNA and tracrRNA, Integrated DNA Technologies Inc.) in Opti-MEM (Thermo Fisher Scientific Inc., MA, USA). crRNA was designed to target the tyrosinase gene in C57BL/6 mice (5′-GGGTGGATGACCGTGAGTCC-3′)^{32 (link)}. The nuclease solution (5 μl) was placed between metal plates of electrodes with a gap of 1 mm on a glass slide (CUY501P1-1.5, NEPA GENE Co. Ltd.). Embryos were placed in a line between electrodes. The poring pulse was set to voltage: 40 V, pulse length: 2.0 ms, pulse interval: 50 ms, number of pulses: 4, decay rate: 10%, polarity: + . The transfer pulse was set to a voltage: 15 V, pulse length: 50 ms, pulse interval: 50 ms, number of pulses: 5, decay rate: 40%, polarity: + / − . The genome editing rates of the offspring were estimated using the eye color difference (white: successful genome edition; black: not successful).