An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells

Abstract

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device’s output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is loading of transcriptional and translational resources, which can induce coupling among otherwise independently-regulated genes. Here, we quantify the effects of resource loading in engineered mammalian genetic systems and develop an endoribonuclease-based feedforward controller that can adapt the expression level of a gene of interest to significant resource loading in mammalian cells. Near-perfect adaptation to resource loads is facilitated by high production and catalytic rates of the endoribonuclease. Our design is portable across cell lines and enables predictable tuning of controller function. Ultimately, our controller is a general-purpose device for predictable, robust, and context-independent control of gene expression.

A call for caution in analysing mammalian co-transfection experiments and implications of resource competition in data misinterpretation

“…More recently, resource competition in mammalian cells has been quantified and shown to undermine the performance of genetic circuits, prompting a deeper investigation of the issue 6 – 8 . Frei et al demonstrated that, at very least, both transcription and translation are affected by intracellular resource availability 7 .…”

“…Hence, when one module is repressed by Dox induction, more resources become available for the other one to be expressed. In a paper by Jones et al 8 resources limitation in the context of transient transfections was probed using a genetic model system in which the GAL4 DNA binding domain (DBD) was fused to activation domains of different strength. By looking at the expression of a co-transfected constitutive reporter driven by different promoters, the authors proved that the stronger the activation by the synthetic transcription factor, the more resource demanding the synthetic device, and the sharper the decrease in the reporter expression.…”

“…C Resource loading following co-transfection can be avoided by using external reporters to control for inter-experimental variability in transfection efficiency (adapted from Brown et al 31 ). D Incoherent feed-forward loops (iFFLs), where cellular resources act as the input regulating the expression of both a mitigator and a mitigator-repressed output gene, are an effective way to buffer the output gene expression to fluctuations in the pool of cellular resources (adapted from Jones et al 8 and Lillaci et al 6 ). …”

“…Previously, this strategy has been explored in microbial systems 32 – 35 . In mammalian cells, a method recently proposed is the incoherent feedforward loop (iFFL) circuit topology, which represents a core motif in biology and has been often used in synthetic circuits for its adaptation properties 6 , 8 . In iFFLs an input A regulates the expression of both an intermediate gene B (resource mitigator) and an output gene C, which is also negatively regulated by B (Fig.…”

“…2D ). Jones et al designed an endoribonuclease-based incoherent feed-forward loop (iFFL) where cellular resources act as the input A, regulating the Cas6 (B)-driven repression of the output gene C 8 . When cellular resource availability decreases (e.g., due the presence of competing modules), the expression of Cas6 diminishes, increasing the expression of the output gene.…”