Combining positive and negative regulation for modular and robust biomolecular control architectures

Engineered biotechnologies are powered by synthetic gene regulation and control systems, known as genetic circuits, which must be modular and robust to disturbances if they are to perform reliably. An emerging family of regulatory mechanisms is mediated by clustered interspaced palindromic repeats (CRISPR) that can both interfere with (downregulate) or activate (upregulate) a given gene’s expression. However, all CRSIPR regulation relies on a shared resource pool of dCas9 proteins. Hence, a circuit’s components can indirectly affect one another via resource competition – even without any intended interactions between them – which compromises the modularity of synthetic biological designs. Using a resourceaware model of CRISPR regulation, we find that circuit modules which simultaneously subject a gene to CRISPR interference and activation are rendered robust to resource competition crosstalk. Evaluating this architecture’s simulated performance, we identify the scenarios where it can be advantageous over the extant resource competition mitigation strategies. We then consider different feedback architectures to demonstrate that combining opposite regulatory interactions overcomes the trade-off in robustness to perturbations of different nature. The motif of combined positive and negative regulation may therefore give rise to more robust and modular biomolecular controllers, as well as hint at the characteristics of natural systems that possess it.