Characterization and mitigation of gene expression burden in mammalian cells

Abstract

Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

dCas9 regulator to neutralize competition in CRISPRi circuits

“…The general problem of resource competition in genetic circuits has been widely studied in the context of competition for translation resources in bacteria 7,20,21 and for transcription resources in mammalian cells 22,23 . In particular, studies in bacteria have shown that the effects of ribosome competition on the I/O response of a genetic circuit can be very subtle, yet dramatic, as the inner circuit's modules compete with one another for ribosomes 20 .…”

Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles

“…The response of expressed genes to their extracellular (or intracellular) inputs are often stochastic and thus imprecise across individual cells 75,76 . In addition, the intracellular context affects the level of gene expression induced by signaling 40,41 , due to factors such as off-target interactions among engineered genes 58 or resource competition among genes 73,77,78 . To remedy these issues and enable the construction of genetic devices that enforce precise and robust signaling responses across cells, we developed a phosphorylation-based negative feedback controller of gene expression (Figure 5a).…”

Gene switch for l‐glucose‐induced biopharmaceutical production in mammalian cells

“…Intriguingly, only the use of the weaker P PGK promoter, but not the stronger P hEF1α promoter, afforded a functional gene switch. We attribute this effect to competition between transgenes for the cellular transcriptional and translational resources (Frei et al, 2020); the stronger P EF1a promoter might sequester resources that would otherwise be available for expression of the reporter gene. Additional expression of the transporter SMIT1 increased the fold switching in CHO‐K1 cells ( p = .0397), in accordance with the anticipated facilitation of l ‐glucose transport across the plasma membrane.…”