Abstract
This study designed three sgRNAs (sgRNA139, sgRNA128, and sgRNA109) targeting the prolactin gene receptor (PRLR) in fetal cattle, utilized Cas9 to cleave endogenous DNA, and screened stable cell lines for somatic cell nuclear transfer experiments to investigate the impact of different editing sites on embryonic development. The results showed that sgRNA139 had the highest cleavage efficiency (Fcut = 0.65, Indels = 42.19%), while sgRNA109 had the lowest (Fcut = 0.45, Indels = 35.31%). No significant differences were observed in cell growth status after electroporation (p > 0.05), and the transfection efficiency exceeded 90% after five days of culture. In the evaluation of key embryonic development indicators, sgRNA109 significantly reduced the cleavage rate and blastocyst rate (p < 0.01), whereas sgRNA139 showed no significant effect on the cleavage rate (p > 0.05), but its blastocyst rate was slightly lower than that of the control group (p > 0.05). This study demonstrates that highly specific sgRNAs and stable edited cell lines used as donor cells can significantly regulate the later stages of embryonic development. This study not only provides new experimental evidence for the functional study of the PRLR but also lays an important theoretical foundation for the innovation of molecular breeding technologies in dairy cattle.