Regulated Expression of sgRNAs Tunes CRISPRi in E-coli
Regulated Expression of sgRNAs Tunes CRISPRi in E-coli
复制标题
DOI：
10.1002/biot.201800069
复制
发表时间：
2018-09-01
影响因子：
4.7
通讯作者：
Carothers, James M.
中科院分区：
文献类型：
--
作者：
Fontana, Jason;Dong, Chen;Carothers, James M.
实现动态控制多基因程序的方法可以扩展设计代谢的能力，以有效地生产高价值化合物。这项工作探讨了是否可以通过调控CRISPRi机制的表达来调节大肠杆菌中的CRISPRi抑制。当dCas9不受限制时，仅sgRNA表达的变化就可以导致CRISPRi的抑制水平从5到300倍不等。在2.5倍的范围内滴定sgRNA表达导致报告基因表达变化16倍。许多不同种类的基因控制器可以产生2.5倍的转录差异，这表明它们可能被整合到动态调节的CRISPRi电路中。最后，CRISPRi不能通过在生长阶段后期表达竞争的sgRNA而逆转长达12小时，这表明CRISPR-Cas:DNA相互作用可以在体内持续存在。总的来说，这些结果确定了通过调节sgRNA表达来调节CRISPRi抑制的遗传结构，并表明针对多个基因的动态调节CRISPRi系统可能是可以实现的。
Methods for implementing dynamically-controlled multi-gene programs could expand capabilities to engineer metabolism for efficiently producing high-value compounds. This work explores whether CRISPRi repression can be tuned in E. coli through the regulated expression of the CRISPRi machinery. When dCas9 is not limiting, variations in sgRNA expression alone can lead to CRISPRi repression levels ranging from 5- to 300-fold. Titrating sgRNA expression over a 2.5-fold range results in 16-fold changes in reporter gene expression. Many different classes of genetic controllers can generate 2.5-fold differences in transcription, suggesting they may be integrated into dynamically-regulated CRISPRi circuits. Finally, CRISPRi cannot be reversed for up to 12 hours by expressing a competing sgRNA later in the growth phase, indicating that CRISPR-Cas:DNA interactions can be persistent in vivo. Collectively, these results identify genetic architectures for tuning CRISPRi repression through regulated sgRNA expression and suggest that dynamically-regulated CRISPRi systems targeting multiple genes may be within reach.