Plug-and-Play or Plug-and-Pray? Impact of random versus targeted integration on the linearity and orthogonality of synthetic gene circuits in CHO cells

Predictable and tuneable gene expression is essential for advanced mammalian cell engineering yet remains challenged by genomic position effects and copy number variability. This study systematically compares random integration (RI) and targeted integration (TI) of two synthetic lineariser gene circuits, TetR/eGFP_Lin and PhlFd/mCherry_Lin, in CHO-S cells. Both circuits exhibited strong linearity and orthogonality across integration strategies, but integration method and repressor choice produced distinct performance trade-offs. RI increased achievable dynamic range for PhlFd but increased basal leakiness for TetR, whereas TI, via a dual-landing-pad system, constrained copy number, and reduced basal expression and PhlFd expression variability. These findings highlight integration context and circuit architecture as key design parameters informing rational design of synthetic regulators for biopharmaceutical applications.