Objective Plasmids construction and knocking out HIF-1α gene of NSF cell line by CRISPR/Cas9 genomic editing technology, providing an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia related diseases in naked mole-rats. Methods Design four pairs of sgRNA sequences which targeted exon 1-4 of NSF HIF-1α gene and successfully construct an expression plasmid. The plasmid with optimal sgRNA was identified and transfected into 293T cells, and the supernatant was used for detecting the virus titer. Furthermore, lentivirus particles carring sgRNAs of HIF-1α infected NSF cells which have been infected with Cas9 lentivirus particles based on the protocol of manufacture previously. After drug screening of post transfection, fluorescence signals were observed under fluorescent microscope, and the expression of HIF-1α in NSF cells were detected by Western blot and T7E1 enzyme. Results The Sanger sequencing results showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors, demonstrating that the recombinant plasmid used for the transfection were successfully constructed;The T7E1 digestion experiment successfully removed 3 bands, the target efficiency of sgRNA was 54%, and the Western blotting results showed that the HIF-1α gene was successfully knocked out and the protein level was significantly reduced in NSF cells of naked mole rats (P=0.0019). Moreover, no obvious changes in the morphology of HIF-1α knockout cells were found under the microscope, and gene knockout had no obvious effect on cell proliferation. Conclusion The HIF-1α knockout cell line was successfully constructed using CRISPR/Cas9 technology, which will provide experimental basis for the further study of the HIF-1α biological function. Furthermore, it will be beneficial for the study of the mechanism of hypoxia tolerance in naked mole mice and provide theoretical foundation for the prevention and treatment of hypoxia related diseases.