Abstract
Methods to regulate gene expression programs in bacterial cells are limited by the absence of effective gene activators. To address this challenge, we have developed synthetic bacterial transcriptional activators in E. coli by linking activation domains to programmable CRISPR-Cas DNA binding domains. Effective gene activation requires target sites situated in a narrow region just upstream of the transcription start site, in sharp contrast to the relatively flexible target site requirements for gene activation in eukaryotic cells. Together with existing tools for CRISPRi gene repression, these bacterial activators enable programmable control over multiple genes with simultaneous activation and repression. Further, the entire gene expression program can be switched on by inducing expression of the CRISPR-Cas system. This work will provide a foundation for engineering synthetic bacterial cellular devices with applications including diagnostics, therapeutics, and industrial biosynthesis.
Highlights
Synthetic control of gene expression has recently become much more straightforward with the emergence of programmable transcription factors using the CRISPR-Cas system (Fig. 1)
To recruit transcriptional activators to the CRISPR-Cas system, we used guide RNAs (gRNAs) that are extended with hairpin sequences to recruit RNA binding proteins (RBPs), which are in turn fused to candidate activators (Fig. 1a)[10]
SoxS may be an effective activator in part because it interacts with the C-terminal domain of RpoA, which is connected to RNA polymerase by a relatively flexible tether[12,43]
Summary
Synthetic control of gene expression has recently become much more straightforward with the emergence of programmable transcription factors using the CRISPR-Cas system (Fig. 1). A catalytically-inactive Cas[9] (dCas9) protein can be used to target specific DNA sequences with guide RNAs (gRNAs) that recognize their targets based on predictable Watson-Crick base pairing. This approach can be used to repress genes by physically blocking RNA polymerase (CRISPR interference or CRISPRi)[6,7]. It is possible to fuse RNA polymerase subunits directly to DNA binding domains to activate transcription[12,13,14]. We show that bacterial CRISPRa can be used to increase the output of a heterologous ethanol biosynthesis pathway These results provide a framework for implementing CRISPRa in bacteria with a wide variety of potential applications. Because SoxS interacts with a highly conserved site on RNA polymerase, our bacterial CRISPRa toolkit may be portable to a broad range of bacterial species
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
