In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nucleotide guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging ∼9 generations, a dCas12a oscillator design with 40-nucleotide CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ hours at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally-independent behavior, as the RNAs control each others’ transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.

中文翻译：

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使用失活的CRISPR-Cas来实现翻译独立的基于RNA的合成振荡器

在合成电路中，CRISPR-Cas系统已被有效地用于从初始状态到最终状态的端点更改，例如在逻辑门中。在这里，我们使用失活的Cas9（dCas9）和失活的Cas12a（dCas12a）来构建动态RNA环形振荡器，该环形振荡器在细菌细胞中随时间在状态之间连续循环。尽管我们使用103个核苷酸的引导RNA的dCas9电路显示出不规则的波动，峰间周期长度平均分布了约9代，但带有40个核苷酸的CRISPR RNA的dCas12a振荡器设计却表现得更好，强烈抑制了状态，明显的自相关函数峰值和约7.5代的平均峰峰值周期长度。连同自由运行的振荡器电路，我们用可诱导的RNA步骤测量开环系统中的阻抑反应时间，以与振荡器周期时间进行比较。我们使用微流控设备在单细胞水平上跟踪数千个细胞24小时以上。在创建具有几乎不依赖翻译的行为的电路时，由于RNA控制着彼此的转录，我们提出了一种合成振荡器的可能性，该振荡器可以在许多生物中普遍存在并且可以很容易地连接用于转录控制。