A Tunable Long\nDuration Pulse Generation Circuit in\nMammalian Cells

Pulse generator circuits based on incoherent feed-forward\nlogic\nhave been developed in bacterial, yeast, and mammalian systems but\nare typically limited to production of short pulses lasting less than\n1 day. To generate longer-lasting pulses, we introduce a feedback-based\ntopology that induces multiday pulsatile gene expression with tunable\nduration and amplitude in mammalian cells. We constructed the circuit\nusing the PERSIST platform, which consists of entirely post-transcriptional\nlogic, because our experience suggests that this approach may attenuate\nlong-term epigenetic silencing. To enable external regulation of PERSIST\nregulatory elements, we engineered inducer-stabilized CRISPR endoRNases\nthat respond to FDA-approved drugs, generating small molecule responses\nwith greater than 20-fold change. These inducer-responsive proteins\nwere connected to a two-state cross-repression positive feedback topology\nto generate the pulse generator circuit architecture. We then optimized\ncircuit design through chromosomal integration of circuit components\nat varying stoichiometries, resulting in a small library of circuits\ndisplaying tunable pulses lasting between two and 6 days in response\nto a single 24 h input of inducer. We expect that the small molecule-stabilized\nPERSIST proteins developed will serve as valuable components in the\ntoolbox for post-transcriptional gene circuit development and that\ntunable post-transcriptional pulse generator circuits in mammalian\ncells will enable study of endogenous hysteretic gene networks and\nsupport advances in cell therapies and organoid engineering.