【Abstract】 Objective To establish a Nestin-Cre site-specific knock-in rat model using CRISPR/Cas9 technology, thereby providing a reliable and efficient genetic tool for research on nervous system development and disease mechanisms. Methods Specific sgRNAs targeting downstream region of Nestin exon 5 were designed and a homologous recombination donor plasmid containing an IRES-Cre expression cassette was constructed. The CRISPR/Cas9 system components and the donor vector were co-microinjected into rat zygotes, followed by embryo transfer to generate F0 generation rats. Positive individuals with correct knock-in were identified by PCR and Sanger sequencing, then mated with wild-type rats to obtain F1 offspring for validation of germline transmission stability. To evaluate the tissue-specific activity of Cre recombinase, F1 knock-in rats were crossed with Rosa26imCherry fluorescent reporter rats. The expression of mCherry in the brain, spinal cord, and various peripheral organs of the offspring was systematically examined using immunofluorescence technology. Results High-efficiency sgRNAs and donor plasmids were successfully prepared. Through microinjection and embryo transfer, F0 founders with precise knock-in were obtained, and the genetic modification was confirmed to be stably transmitted to the F1 generation. Results of tissue immunofluorescence revealed high-level expression of mCherry in the central nervous system (brain and spinal cord) of the hybrid offspring. Scattered fluorescence signals were observed in peripheral tissues, including the heart, liver, spleen, lung, kidney, ovary, and muscle, consistent with the endogenous developmental expression profile of Nestin. Significant co-localization of mCherry with the mature neuronal marker NeuN in brain tissues indicated that the Cre recombinase activity possesses clear neuronal lineage specificity.? Conclusion A Nestin-Cre knock-in rat model was successfully established. This model achieves efficient and specific expression of Cre recombinase within the central nervous system, with a stably inheritable modification site. It provides a valuable tool strain for conducting neuron-specific gene function studies in vivo.