Abstract
Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on σ70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ54-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ54-dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications.
Highlights
Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression
It has been proven that the bacterial Enhancer Binding Proteins” (bEBPs) PspF from E. coli has a highly modular structure—a truncated PspF remained as a functional activation domain in a three-hybrid system in vitro[20,21]
3 bp downstream of the native upstream activating sequence (UAS) were mutated to an NGG PAM, which could be targeted by a dCas9–sgRNA complex
Summary
Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. Our CRISPRa device can activate σ54-dependent promoters with biotechnology relevance in non-model bacteria It supports orthogonal gene regulation on multiple levels. A σ54-activator binds to an upstream activating sequence (UAS) and performs long-distance regulation through a local DNA loop, and catalyzes ATP hydrolysis to unlock the complex for transcription initiation This mechanism is similar to transcriptional activation involving the eukaryotic RNA polymerase II in several ways and is known as “eukaryotelike” regulation in bacteria[13,14,15,16,17,18]. We set up an efficient profile scanning platform for metabolic pathways, in which the sgRNA library and its encoded transcriptional activation profiles do not need to be rebuilt and can be applied to different pathways Those profiles, when applied to multi-gene expression, show good stability and durability. This CRISPRa device enriches the genetic regulation toolbox, and supports real-life application scenarios by its high performance
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
