Kim Seong Keun, Woo Seung-Gyun, Park Jun-Hong, Lee Seung-Goo, Lee Dae-Hee
Synthetic Biology Research Center and The K-Biofoundry, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA.
J Biol Eng. 2025 Jun 6;19(1):56. doi: 10.1186/s13036-025-00526-8.
Precise and dynamic transcriptional regulation is a cornerstone of synthetic biology, enabling the construction of robust genetic circuits and programmable cellular systems. However, existing regulatory tools are often limited by issues such as leaky transcription and insufficient tunability, particularly in high-expression or complex genetic contexts. This study aimed to develop a CRISPRi-aided genetic switch platform that overcomes these limitations and expands the functionality of transcriptional regulation tools in synthetic biology.
We established a versatile CRISPRi-aided genetic switch platform by integrating transcription factor-based biosensors with the Type V-A FnCas12a CRISPR system. Exploiting the RNase activity of FndCas12a, this system processes CRISPR RNAs (crRNAs) directly from biosensor-responsive mRNA transcripts, enabling precise, signal-dependent transcriptional regulation. To mitigate basal transcription and enhance regulatory precision, transcriptional terminator filters were incorporated, reducing leaky expression and increasing the dynamic range of target gene regulation. The platform demonstrated exceptional adaptability across diverse applications, including ligand-inducible genetic switches for transcriptional control, signal amplification circuits for enhanced output, and metabolic genetic switches for pathway reprogramming. Notably, the metabolic genetic switch dynamically repressed the endogenous gapA gene while compensating with orthologous gapC expression, effectively redirecting metabolic flux to balance cell growth.
The CRISPRi-aided genetic switch provides a powerful and flexible toolkit for synthetic biology, addressing the limitations of existing systems. By enabling precise and tunable transcriptional regulation, it offers robust solutions for a wide array of biotechnological applications, including pathway engineering and synthetic gene networks.
精确且动态的转录调控是合成生物学的基石，能够构建稳健的遗传电路和可编程细胞系统。然而，现有的调控工具常常受到诸如转录泄漏和可调性不足等问题的限制，特别是在高表达或复杂的遗传背景下。本研究旨在开发一种CRISPRi辅助的遗传开关平台，以克服这些限制并扩展合成生物学中转录调控工具的功能。
我们通过将基于转录因子的生物传感器与V-A型FnCas12a CRISPR系统整合，建立了一个通用的CRISPRi辅助遗传开关平台。利用FnCas12a的核糖核酸酶活性，该系统直接从生物传感器响应的mRNA转录本中加工CRISPR RNA（crRNA），实现精确的、信号依赖的转录调控。为了减轻基础转录并提高调控精度，引入了转录终止子过滤器，减少了泄漏表达并增加了靶基因调控的动态范围。该平台在各种应用中表现出卓越的适应性，包括用于转录控制的配体诱导遗传开关、用于增强输出的信号放大电路以及用于途径重编程的代谢遗传开关。值得注意的是，代谢遗传开关动态抑制内源性gapA基因，同时通过同源gapC表达进行补偿，有效地重定向代谢通量以平衡细胞生长。
CRISPRi辅助遗传开关为合成生物学提供了一个强大且灵活的工具包，解决了现有系统的局限性。通过实现精确且可调的转录调控，它为广泛的生物技术应用提供了稳健的解决方案，包括途径工程和合成基因网络。
Zotero 插件
