基于非相干前馈逻辑的脉冲发生器电路已在细菌、酵母和哺乳动物系统中开发出来，但通常仅限于产生持续时间少于 1 天的短脉冲。为了产生更持久的脉冲，我们引入了一种基于反馈的拓扑结构，可在哺乳动物细胞中诱导持续时间和幅度可调的多日脉冲基因表达。我们使用 PERSIST 平台构建了该电路，该平台完全由转录后逻辑组成，因为我们的经验表明这种方法可能会减弱长期表观遗传沉默。为了实现 PERSIST 调控元件的外部调控，我们设计了诱导剂稳定的 CRISPR 内切 RNA 酶，该酶可对 FDA 批准的药物做出反应，产生变化超过 20 倍的小分子反应。这些诱导物响应蛋白连接到二态交叉抑制正反馈拓扑，以生成脉冲发生器电路架构。然后，我们通过不同化学计量的电路组件的染色体集成来优化电路设计，从而形成一个小型电路库，可响应感应器的单次 24 小时输入，显示持续 2 至 6 天的可调脉冲。我们期望开发的小分子稳定的 PERSIST 蛋白将作为转录后基因电路开发工具箱中的有价值的组成部分，并且哺乳动物细胞中可调节的转录后脉冲发生器电路将能够研究内源性滞后基因网络并支持在细胞疗法和类器官工程。

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.