Equalizer Circuits Provide Constant Gene Expression Regardless of Plasmid Copy Number in Mammalian Cells

Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of plasmid copy numbers per cell, and consequently, high expression heterogeneity. High expression heterogeneity can make genetic circuits less predictable and unreliable. Here, we report plasmid-based synthetic circuits – Equalizers – that buffer copy number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. We demonstrate that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable a rapid generation of extrachromosomal cell lines with stable and uniform expression for over two months. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in individual cells.