Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the 'repressilator' can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over the time course of several days. Through statistical analysis of the single cell data, the potential for the circuit to act as a synthetic pacemaker for cellular processes is evaluated. The use of CRISPRi in the context of an oscillator circuit is found to have profound effects on its dynamics. Specifically, irreversible binding of dCas9-sgRNA appears to prolong the period of the oscillator. Further, we demonstrate that the oscillator affects cellular growth, leading to variations in growth rate with the oscillator's frequency.

中文翻译：

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具有包含dCas9-sgRNA的混合反馈回路的合成细菌时钟的单细胞表征

在整个生命的各个王国中，广泛发现了在基因表达活动中产生振荡的遗传网络。振荡动力学促进了细胞和生物体内新陈代谢和生长过程的时间协调，以及这种过程与环境中周期性发生变化的同步。诸如“再加压器”之类的合成振荡器基因电路可以在细菌中执行类似的功能。直到最近，此类电路主要基于相对较少的一组特征明确的转录阻遏物和激活物。CRISPR干扰（CRISPRi）提供了一种有前途的，可进行序列编程的基因调节方法，CRISPR干扰（CRISPRi）可以转录抑制短链RNA分子指导的几乎任意基因靶标。为了证明在动态基因回路的背景下使用CRISPRi，我们在这里用RNA引导的dCas9蛋白替换了再加压回路的一个节点。使用微流体反应器中的单细胞实验，我们表明该系统在多个时期以及几天的时间过程中显示出强大的弛豫振荡。通过对单细胞数据的统计分析，可以评估电路充当细胞过程的合成起搏器的潜力。发现在振荡器电路中使用CRISPRi对其动力学产生深远影响。具体而言，dCas9-sgRNA的不可逆结合似乎延长了振荡器的周期。此外，我们证明了振荡器会影响细胞的生长，从而导致振荡器的生长速率发生变化。