Coupling Shared and Tunable Negative Competition Against Winner-Take-All Resource Competition Via CRISPRi Moieties

Competition for cellular resources often indirectly creates unintended connections between otherwise independent genetic modules, leading to loss of modularity and impairment of intended circuit function. Both global and local negative feedback control strategies have been widely-used as attempts to mitigate the undesired effects of resource competition. However, these controllers demonstrate limited tunability and scalability with increasing circuit complexity. Our previous work attempting to achieve two successive cell fate transitions via cascading bistable switches demonstrates how resource competition can lead to winner-take-all (WTA) dynamics that deviate from the intended behavior. Here, we attempt to remediate these issues by synthetically introducing a shared and tunable system of negatively competitive regulation (NCR), incorporating repressive CRISPR moieties to free up cellular resources from the winner module to a degree proportional to its activity. This system punishes transcriptional modules that take up more than their fair share of resources while having minimal effect on modules operating within normal activity ranges. We compare this novel mode of regulation to global and local negative feedback controllers and demonstrate the significantly increased efficacy of controlling WTA resource competition NCR can achieve. Thus, we provide an alternative strategy for controlling resource competition.