A Tunable Long Duration Pulse Generation Circuit in Mammalian Cells

Pulse generator circuits based on incoherent feed-forward logic have been developed in bacterial, yeast, and mammalian systems but are typically limited to production of short pulses lasting less than 1 day. To generate longer-lasting pulses, we introduce a feedback-based topology that induces multiday pulsatile gene expression with tunable duration and amplitude in mammalian cells. We constructed the circuit using the PERSIST platform, which consists of entirely post-transcriptional logic, because our experience suggests that this approach may attenuate long-term epigenetic silencing. To enable external regulation of PERSIST regulatory elements, we engineered inducer-stabilized CRISPR endoRNases that respond to FDA-approved drugs, generating small molecule responses with greater than 20-fold change. These inducer-responsive proteins were connected to a two-state cross-repression positive feedback topology to generate the pulse generator circuit architecture. We then optimized circuit design through chromosomal integration of circuit components at varying stoichiometries, resulting in a small library of circuits displaying tunable pulses lasting between two and 6 days in response to a single 24 h input of inducer. We expect that the small molecule-stabilized PERSIST proteins developed will serve as valuable components in the toolbox for post-transcriptional gene circuit development and that tunable post-transcriptional pulse generator circuits in mammalian cells will enable study of endogenous hysteretic gene networks and support advances in cell therapies and organoid engineering.