Department of Fundamental Microbiology, University of Lausanne, Biophore Building, 1015, Lausanne, Switzerland.
Department of Biosystems Science and Engineering, ETH Zurich and SIB Swiss Institute of Bioinformatics, Basel, Switzerland.
Nat Commun. 2020 Jun 2;11(1):2746. doi: 10.1038/s41467-020-16574-1.
Gene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable synthetic circuit construction. Here we use CRISPRi to build a synthetic oscillator ("CRISPRlator"), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allow us to achieve robust circuit behaviors in Escherichia coli populations. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.
基于 CRISPRi（成簇规律间隔短回文重复干扰）的基因表达控制已经成为在原核生物和真核生物中构建合成基因回路的有力工具；然而，其缺乏协同性被指出是动态或多稳态合成电路构建的潜在障碍。在这里，我们使用 CRISPRi 构建了一个合成振荡器（“CRISPRlator”）、双稳态网络（toggle switch）和条纹模式形成非相干前馈环（IFFL）。我们的电路设计旨在具有高可预测性和正交性，以及低代谢负担和上下文相关性，使我们能够在大肠杆菌群体中实现稳健的电路行为。数学建模表明，CRISPRi 中的非特异性结合对于建立多稳态是必不可少的。我们的工作证明了 CRISPRi 在合成电路中的广泛适用性，并为未来通过 CRISPR 技术的优势来设计更复杂的合成网络铺平了道路。
