CRISPR-Based Genetic Modification of Embryos

MII oocytes were collected as described above. The RNP complex was co-injected with sperm during ICSI^{38 (link)}. MII oocytes and sperm were washed in a drop of 200 ng/µl RNP complex in opti-MEM (Invitrogen), and ICSI was performed in the drop. Electroporation of the RNP complex into ICSI embryos was performed using a Super Electroporator NEPA21 (Nepa Gene). In brief, 10 min after ICSI, zygotes were placed in the glass chamber between 1-mm-gap platinum electrodes (Nepa Gene); this chamber was filled with 6 µl of opti-MEM containing 200 ng/µl RNP complex^{39 (link)}. The poring pulse parameters were as follows: voltage, 40 V; pulse width, 3.5 ms; pulse interval, 50 ms; and number of pulses, +4. The transfer pulse parameters were as follows: voltage, 5 V; pulse width, 50 ms; pulse interval, 50 ms; and number of pulses, ±5.