Dual-mode CRISPRa/i for genome-scale metabolic rewiring in Escherichia coli.

CRISPR (clustered regularly interspaced palindromic repeats)-mediated transcriptional regulation is a powerful and programmable approach for controlling gene expression. While CRISPR-based gene repression is well established in bacteria, simultaneous activation and repression remain challenging due to the limited availability of effective bacterial activation domains. Here, we provide an efficient dual-mode CRISPR activation and interference (CRISPRa/i) system that integrates an evolved protospacer adjacent motif (PAM)-flexible dxCas9 with an engineered Escherichia coli cAMP receptor protein (CRP). Through systematic optimization of the CRP domains and linkers, we developed a versatile effector capable of precise gene expression control when combined with dxCas9. Our dxCas9-CRP system demonstrated robust activation of upstream regulatory regions and effective repression of coding sequences, enabling targeted and programmable gene regulation. Using dual-fluorescent reporters, we validated the ability of this system to concurrently regulate multiple genes. Furthermore, with pooled guide RNA libraries, we applied the dxCas9-CRP system to increase violacein production in E. coli via genome-scale activation and repression in a coordinated manner, successfully identifying key regulatory targets that significantly increase production. Overall, this dual-mode CRISPRa/i system advances the potential for bacterial metabolic pathway rewiring, providing precise and flexible control for a wide range of biotechnological applications.