<p>This study aims to explore and optimize the conditions for in vivo transfection and gene editing, emphasizing their potential applications in the treatment of infertility in mice. Our findings indicate that physical transfection can be effective in the dynamic fluid environment of mouse seminiferous tubules, with electroporation achieving transfection in multilayered cell tissues. Using the <i>mTmG</i> fluorescence reporter system, we visually assessed the efficiency of electroporation-based transfection and observed stable gene editing outcomes across different individuals. Additionally, we achieved effective transfection of germ cells in vivo for the clinical application of gene tools. We further investigated the impact of various delivery methods and molecular methods on transfection efficacy, revealing that RNP technology is adaptable and efficient in vivo, particularly in the context of treating hereditary diseases. We attempted to leverage gene editing techniques to address spermatogenesis blockage at different stages in <i>Ythdc2</i>-KO and <i>CK137956</i>-KO mice. While we did not succeed in rescuing spermatogenic blockage in <i>Ythdc2</i> KO mice, the treatment of <i>CK137956</i> KO mice yielded significant physiological responses. These results could be beneficial for the optimization of in vivo gene editing technologies for clinical applications.</p> Graphical Abstract <p></p>