Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.
Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.
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DOI：
10.1093/nar/gkw056
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发表时间：
2016-03-18
影响因子：
14.9
通讯作者：
Moon TS
中科院分区：
文献类型：
--
作者：
Lee YJ;Hoynes-O'Connor A;Leong MC;Moon TS
合成生物学的一个中心目标是通过可预测地控制基因表达来实现不同的细胞功能。虽然研究更多地集中在蛋白质调节剂而不是RNA调节剂上，但我们对RNA折叠和功能的理解的最新进展促使了RNA调节剂的使用。RNA调节剂提供了一个优势，因为它们比蛋白质调节剂更容易设计和工程化，对细胞的负担可能更低，并且是高度正交的。在这里，我们将来自化脓性链球菌的CRISPR系统与大肠杆菌菌株中的合成反义RNA（asRNA）结合起来，以可编程的方式抑制或去抑制靶基因。具体来说，我们首次证明了CRISPR系统抑制的基因靶标可以通过表达一种asRNA来解除抑制，这种asRNA可以螯合一种小向导RNA（sgRNA）。此外，我们证明，通过设计靶向sgRNA不同区域的asRNA和通过改变sgRNA-asRNA复合物的杂交自由能，可以实现可调水平的去阻遏（高达95%）。这种新系统，我们称之为CRISPR和asRNA组合系统，可以用于同时可逆地抑制或去抑制多个靶基因，从而实现细胞功能的合理重编程。
A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.