Objective To verify the feasibility of Cas9 microinjection in frozen-thawed pronuclear embryos, based on the model of pronuclear embryos of C57BL/6J mice by in vitro fertilization. Methods After fertilized mouse pronuclear embryos cultured in vitro, one-cell and 2-cell embryos were frozen using EFS20/40 cryopreservation tube. The next day recovered and then cultured. The recovery rate and survival rate of the one-cell and 2-cell embryos were compared. The frozen-thawed and fresh pronuclear embryos were injected with Cas9 mixture and injection buffer into the cytoplasm, and then cultivated to 2-cell embryos,and the survival rate and development rate of the 2-cell embryos were compared. Results The recovery rate of frozen-thawed one-cell embryos was 92.5%, the survival rate was 92.8%, the recovery rate of 2-cell embryos was 90.5% and the survival rate was 95.8%, showing no significant difference. Furthermore, the survival rate of fresh one-cell embryos after Cas9 injection was 92.7%, the survival rate of one-cell embryos of the blank group was 97.5%. While the survival rate of Cas9 injected frozen-thawed one-cell embryos was 82.6%, and that of the blank group was 92%,showing a significant difference between the frozen-thawed injected group and other groups(P<0.05).The development rate of 2-cell embryos after Cas9 injection was not significantly different. Conclusions Frozen-thawed pronuclear embryos can be used for Cas9 microinjection.