PDF(Pubmed)
Abstract：
Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.
摘要：
动态,多输入基因调控网络(GRN)在自然界中普遍存在。基于CRISPR的多层遗传电路对于构建类似于天然存在的生物系统中发现的GRN具有很大的希望。我们开发了一种方法来创建高性能的可激活启动子，宽,和多输入CRISPR激活和干扰(CRISPRa/i)GRN。通过整合基于序列的设计和体内筛选，我们设计可激活的启动子，在基于大肠杆菌的无细胞系统中实现高达1,000倍的动态范围。这些组件使CRISPRaGRN具有六层深和四个分支宽。我们通过将依赖光的EL222光遗传系统的动态范围从6倍提高到34倍，显示了启动子工程工作流程的普遍性。此外,高动态范围启动子使小分子和蛋白质-蛋白质相互作用介导的CRISPRa系统成为可能。我们应用这些工具来构建响应输入的CRISPRa/iGRN，包括反馈回路，逻辑门,多层级联，和动态脉冲调制器。我们的工作为高动态范围可激活启动子的设计提供了一种可推广的方法，并使无细胞系统中的基因调控功能成为可能。
