The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the