Stepwise activation of gene copies results in higher final titers of subclones compared to immediate integration of the full set of active copies.

The increasing demand for production of therapeutic proteins has encouraged both industrial and academic institutions to pursue the development of mammalian expression platforms with high productivities. While protocols for rapid and efficient integration of multiple transgene copies into the genome are available, they require substantial time and resources for screening numerous clones. A contributing factor is the tendency of high producers to disappear from the selected mini-pools due to the stress caused by high productivity without adequate time for adaptation of cellular capacities. Here, we have developed a strategy to stably activate individual copies within an initially repressed multicopy coding cassette harboring 2 GFP-Fc and 2 BFP-Fc genes, each fused to an Fc region for secretion. This toolbox enables gene activation via CRISPR/Cas9-mediated deletion of the repressor elements. Subsequently, producers can be sorted based on increased GFP or BFP fluorescence and assessed by measuring the secreted total Fc protein. We demonstrate that the stepwise activation of initially repressed genes outperforms a control cell line with the same number of genes active from the outset, as evidenced by higher fluorescence signals from GFP and BFP, increased mRNA levels for BFP, GFP, and Fc genes, and enhanced titer of secreted Fc fusion protein. This study demonstrates the ability of cells to adapt to new challenges by modulating both gene expression patterns and channeling of resources to accommodate high production loads.