Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, 98195, USA.
Center for Synthetic Biology, University of Washington, Seattle, WA, 98195, USA.
Biotechnol J. 2018 Sep;13(9):e1800069. doi: 10.1002/biot.201800069. Epub 2018 May 11.
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.
方法实施动态控制多基因程序可以扩大能力工程代谢高效生产高价值的化合物。这项工作探讨是否 CRISPRi 抑制可以通过调控 CRISPRi 机械的表达在大肠杆菌中进行调整。当 dCas9 不是有限的，sgRNA 表达的变化可以导致 CRISPRi 抑制水平从 5 到 300 倍不等。在 2.5 倍的范围内调整 sgRNA 的表达导致报告基因表达的 16 倍变化。许多不同类别的遗传控制器可以产生 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 sgRNA 来逆转，这表明 CRISPR-Cas:DNA 相互作用在体内可能是持久的。总之，这些结果确定了通过调节 sgRNA 表达来调整 CRISPRi 抑制的遗传结构，并表明针对多个基因的动态调节 CRISPRi 系统可能即将实现。
