LAUSR

Abstract 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.