〔Abstract〕 Abstract Objective To construct a human keratinocyte-forming cell line (HaCaT) with stable knockout of the G protein-coupled receptor 107 (GPR107) gene , and to preliminarily investigate the effect of GPR107 deletion on the biological behaviour of HaCaT cells . Methods Using CRISPR/Cas9 gene editing technology , HaCaT cells with knockout of GPR107 gene were constructed and monoclonal cells with GPR107 deletion were obtained by limited di- lution method . Genomic DNA was amplified using Western blot and PCR and sequenced to validate the single-cell clones with knockdown of GPR107 . The cell cycle changes were detected by flow cytometry; cell proliferation was detected by CCK-8 ; apoptosis was detected by flow cytometry; changes in cell differentiation markers were detected by Western blot; cell migration ability was analyzed by cell scratch assay and other methods . Results LentiCas9 - Blast and plenti-guide-RNA-GPR107 plasmids were successfully transfected into HaCaT cells , 21 monoclonal cell lines were obtained by limited dilution , and Western blot showed that the GPR107 expression was significantly re- duced in 8 of them; PCR sequencing of the cellular genome was used , which resulted in the obtainment of C4 and 2D8 GPR107 - / - HaCaT monoclonal cell lines . CCK-8 assay and flow cytometry assay showed that GPR107 gene deletion resulted in G0 G1 phase block , significantly weakened proliferation ability and increased apoptosis level of HaCaT cells . Western blot found that the differentiation of HaCaT cells accelerated after knockdown of GPR107. Additionally the results of the cell scratch assay indicated that the migration ability of HaCaT cells was enhanced af- ter knockdown of GPR107. The results showed that the migration ability of HaCaT cells was enhanced after knock- down of GPR107. Conclusion HaCaT cell line with GPR107 gene deletion is successfully constructed , GPR107 deletion blocks the G0 G1 phase of HaCaT cells , which inhibiting the proliferation of HaCaT cells and promoted ap- optosis , and it was found that the differentiation and migration of HaCaT cells were enhanced after knocking down GPR107.