Construction of a stable expression HEK293T engineered cell line adapted to high-density suspension culture

The HEK293 cell line has received FDA approval and is increasingly used to produce therapeutic glycoproteins . However, the cell line faces the challenge of long-term expression instability. This problem could be solved by specifically integrating exogenous genes into stable sites and developing a HEK293 cell platform to efficiently and stably express recombinant proteins with large-scale suspension culture . The study uses CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE) technologies to integrate a variety of exogenous genes into specific chromosomal sites, such as the hROSA26 site, AAVS1 site, and CCR5 site. We evaluated the stability and expression intensity of exogenous proteins at different insertion sites under suspension culture conditions. A monoclonal cell line expressing enhanced green fluorescent protein (EGFP) is obtained using CRISPR/Cas9-mediated specific integration. The positive monoclonal cell lines express EGFP after 60 consecutive passages, and no significant differences in protein expression titer are observed. These results show that, under suspension culture conditions, the hROSA26 locus could stably express exogenous proteins after 60 generations. HEK293 cell lines integrating HSA (68 kDa) and IFNβ-HSA (89 kDa) genes at the above three sites were obtained respectively using Bxb1-mediated RMCE technology, and batch experiments demonstrated their expression capacity. In summary, a stable and efficient protein expression platform of HEK293 cells is successfully constructed and could be adapted to serum-free suspension culture. • We developed a HEK293T cell platform suitable for suspension culture and stable exogenous protein expression. • We developed a strategy for the efficient construction of HEK293 engineered cell lines. • The EGFP gene has been site-specifically integrated and stably expressed at the hROSA26 site. • Secreted proteins undergo site-specific integration mediated via RMCE.