Construction of a CHO cell line with site-specific integration to stably express exogenous proteins using the CRISPR–Cas9 technique

Chinese hamster ovary (CHO) cells are widely used in biopharmaceuticals because of their high-density suspension culture, high safety, and high similarity between expressed exogenous proteins and natural proteins. However, the level of exogenous protein expression decreases with increasing culture time; this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration. The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology. The results showed that the exogenous proteins enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells. The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages, and no significant differences in expression levels were observed. This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.