The CRISPR-Cas system provides a versatile RNA-guided approach for a broad range of applications. Thanks to advances in RNA synthetic biology, the engineering of guide RNAs (gRNAs) has enabled the conditional control of the CRISPR-Cas system. However, achieving precise regulation of the CRISPR-Cas system for efficient modulation of internal metabolic processes remains challenging. In this work, we developed a robust dCas9 regulator with engineered conditional gRNAs to enable tight control of endogenous genes. Our conditional gRNAs in Escherichia coli can control gene expression upon specific interaction with trigger RNAs with a dynamic range as high as 130-fold, evaluating up to a three-input logic A OR (B AND C). The conditional gRNA-mediated targeting of endogenous metabolic genes, lacZ, malT and poxB, caused differential regulation of growth in Escherichia coli via metabolic flux control. Further, conditional gRNAs could regulate essential cytoskeleton genes, ftsZ and mreB, to control cell filamentation and division. Finally, three types of two-input logic gates could be applied for the conditional control of ftsZ regulation, resulting in morphological changes. The successful operation and application of conditional gRNAs based on programmable RNA interactions suggests that our system could be compatible with other Cas-effectors and implemented in other host organisms.

中文翻译：

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使用可编程、多输入处理 CRISPR 引导 RNA 对内源基因表达进行逻辑调控


CRISPR-Cas 系统为广泛的应用提供了一种多功能的 RNA 引导方法。得益于 RNA 合成生物学的进步，引导 RNA (gRNA) 的工程化使得 CRISPR-Cas 系统的条件控制成为可能。然而，实现 CRISPR-Cas 系统的精确调控以有效调节内部代谢过程仍然具有挑战性。在这项工作中，我们开发了一种强大的 dCas9 调节器，其中包含工程化条件 gRNA，以实现对内源基因的严格控制。我们在大肠杆菌中的条件 gRNA 可以通过与触发 RNA 发生特异性相互作用来控制基因表达，动态范围高达 130 倍，最多可评估三输入逻辑 A OR（B AND C）。 gRNA 介导的条件性靶向内源代谢基因 lacZ、malT 和 poxB，通过代谢通量控制对大肠杆菌的生长产生差异调节。此外，条件 gRNA 可以调节必需的细胞骨架基因 ftsZ 和 mreB，以控制细胞丝状化和分裂。最后，三种类型的二输入逻辑门可用于 ftsZ 调节的条件控制，从而导致形态变化。基于可编程 RNA 相互作用的条件 gRNA 的成功运行和应用表明我们的系统可以与其他 Cas 效应器兼容并在其他宿主生物体中实施。