Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria

TLDR

CRISPR/Cas-based transcriptional regulation is widely used, with CRISPRi achieving high efficiencies in bacteria, but CRISPR activation has been limited by reliance on σ70 promoters. The authors present a eukaryote‑like bacterial CRISPRa system that uses σ54‑dependent promoters to enable long‑range, multi‑input regulation with high dynamic ranges. They demonstrate a reusable scanning platform that optimizes metabolic pathways without library reconstructions. The system activates σ54 promoters in non‑model bacteria, supports orthogonal multi‑level regulation, and, when combined with dxCas9, expands targeting flexibility and dynamic ranges to build cascaded CRISPRa circuits, making it a versatile tool for research and industry.

Abstract

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 σ 70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ 54 -dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ 54 -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.