Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system.
Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system.
复制标题
一种用于细菌基因组一步工程的 CRISPR-Cas9 辅助非同源末端连接策略
DOI：
10.1186/s12934-016-0594-4
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发表时间：
2016-11-15
影响因子：
6.4
通讯作者：
Liang Q
中科院分区：
文献类型：
--
作者：
Chang Y;Su T;Qi Q;Liang Q
规则间隔短回文重复序列干扰（CRISPRi）是近年来发展起来的基因调控的有力工具。在大肠杆菌中，与广泛使用的表达dCas 9作为额外质粒的II型系统相比，内源表达的I型CRISPR系统应易于内部调节而不会引起代谢负担。通过敲除cas 3并激活CRISPR-associated complex for antiviral defense（Cascade）的表达，我们在E.杆菌使用绿色荧光蛋白证明了靶基因从6%下调至82%。柠檬酸合成酶基因（gltA）在TCA循环中的调节影响宿主代谢。通过体内聚-3-羟基丁酸酯（PHB）的积累证实了代谢通量调节的作用。通过调节E.使用工程化的内源性I-E CRISPR系统，我们将代谢流从中心代谢途径重定向到PHB合成途径。本研究表明，内源性I-E型CRISPR-Cas系统是一种简单有效的调节内部代谢途径的方法，可用于产物合成。本文的在线版本（doi：10.1186/s12934-016-0594-4）包含补充材料，可供授权用户使用。
Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid. By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E. coli. Downregulation of the target gene from 6 to 82% was demonstrated using green fluorescent protein. Regulation of the citrate synthase gene (gltA) in the TCA cycle affected host metabolism. The effect of metabolic flux regulation was demonstrated by the poly-3-hydroxbutyrate (PHB) accumulation in vivo. By regulating native gltA in E. coli using an engineered endogenous type I-E CRISPR system, we redirected metabolic flux from the central metabolic pathway to the PHB synthesis pathway. This study demonstrated that the endogenous type I-E CRISPR-Cas system is an easy and effective method for regulating internal metabolic pathways, which is useful for product synthesis. The online version of this article (doi:10.1186/s12934-016-0594-4) contains supplementary material, which is available to authorized users.
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影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
3.2
作者：
Deveau, Helene;Barrangou, Rodolphe;Moineau, Sylvain
通讯作者：
Moineau, Sylvain
影响因子：
14.8
作者：
通讯作者：
--
CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.