Rijal Sadikshya, Standage-Beier Kylie, Zhang Rong, Stone Austin, Youssef Abdelrahman, Wang Xiao, Tian Xiao-Jun
School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona, 85281, United States.
bioRxiv. 2025 May 16:2025.05.15.654351. doi: 10.1101/2025.05.15.654351.
Cellular resource limitations create unintended interactions among synthetic gene circuit modules, compromising circuit modularity. This challenge is particularly pronounced in circuits with positive feedback, where uneven resource allocation can lead to Winner-Takes-All (WTA) behavior, favoring one module at the expense of others. In this study, we experimentally implemented a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) and evaluated its effectiveness in mitigating WTA behavior in two gene circuits: dual self-activation and cascading bistable switch. We chromosomally integrated a tunable dCas9 gene and designed module-specific gRNAs, with each module encoding its own gRNA to self-repress via competition for limited dCas9. This configuration introduces strong negative feedback to the more active module while reallocating resources to the less active one, promoting balanced module activation. Compared to the control group lacking dCas9-mediated repression, the NCR controller significantly increased module coactivation and suppressed WTA behavior. Our quantitative results demonstrate that NCR provides an effective strategy for regulating resource competition and improving the modularity of synthetic gene circuits.
细胞资源限制会在合成基因回路模块之间产生意想不到的相互作用，从而损害回路的模块化。这一挑战在具有正反馈的回路中尤为明显，在这种回路中，资源分配不均会导致胜者通吃（WTA）行为，即以牺牲其他模块为代价来偏袒一个模块。在本研究中，我们使用CRISPR干扰（CRISPRi）实验性地实施了一种负竞争性调节（NCR）控制器，并评估了其在减轻两个基因回路（双自激活回路和级联双稳态开关回路）中的WTA行为方面的有效性。我们将一个可调控的dCas9基因整合到染色体上，并设计了模块特异性的gRNA，每个模块编码自己的gRNA，通过竞争有限的dCas9进行自我抑制。这种配置为更活跃的模块引入了强烈的负反馈，同时将资源重新分配给较不活跃的模块，从而促进模块激活的平衡。与缺乏dCas9介导的抑制作用的对照组相比，NCR控制器显著增加了模块的共激活，并抑制了WTA行为。我们的定量结果表明，NCR为调节资源竞争和提高合成基因回路的模块化提供了一种有效的策略。
