A comprehensive evaluation of stable expression “hot spot” in the ScltI gene of Chinese hamster ovary cells

The Chinese hamster ovary (CHO) cell is the most widely used biopharmaceutical expression system, but its long-term expression is unstable. This issue can be effectively addressed by site-specific integration of exogenous genes into the genome. Therefore, exogenous protein sites with stable expression in the CHO cell genome must be identified. CRISPR/Cas9 technology was used in this study to integrate various exogenous genes into the ScltI site as a “hot spot” at the CHO-K1 cell genome NW_003614095.1, and the stability and adaptability of exogenous genes expressed at the site were investigated. Flow cytometry sorting technology was used to obtain positive monoclonal cell lines that expressed either intracellular protein green fluorescent protein (EGFP) or secretory protein human serum albumin (HSA). For 60 passages, the positive monoclonal cell lines’ cell growth cycles and exogenous protein expression were both observed. The results demonstrated that integrating the gene encoding exogenous proteins into the ScltI site had no effect on cell growth. The fluorescence intensity of EGFP was similar after 60 passages, and the expression of HSA increased slightly. Additionally, the super-monomeric protein VWF hydrolase (ADAMTS13) (190 kDa), human coagulation factor VII (FVII) (55 kDa), and interferon α2b (12 kDa) were integrated into the ScltI site for expression. In conclusion, the site located in the first exon of the ScltI gene within the CHO-K1 cell genome NW_003614095.1 is an ideal “hot spot” for the stable expression of various exogenous proteins. Key points • The site-specific integration strategy of an exogenous gene in CHO cells was established for the ScltI site . • The genes for EGFP and HSA were site-directed integrated and stably expressed at the ScltI site . • The ScltI site fulfills the expression of exogenous proteins of different molecular weight sizes (15–190 kDa) .