Tunable Transcription-Level CRISPR Interference in Vibrio natriegens Using Engineered Single Guide RNAs

Vibrio natriegens, a fast-growing bacterium, has emerged as a promising next-generation microbial platform for microbiology and biological engineering. While an expanding toolkit of genetic parts and genome engineering methods has been established, strategies for precise and predictable control of gene expression remain limited. Here, we report a dCas9-based tunable CRISPR interference (CRISPRi) system that enables multilevel transcriptional regulation in V. natriegens. By engineering the tetraloop and flanking regions of single-guide RNA (sgRNA), we constructed a synthetic sgRNA library that modulates the binding affinity between sgRNA and dCas9. The resulting sgRNA variants exhibited modular repression behavior across multiple protospacer targets. We further demonstrated the utility of this tunable CRISPRi system in metabolic engineering applications by redirecting intracellular carbon flux. Tunable repression of endogenous genes led to a 2.2-fold increase in 3-hydroxypropionic acid (3-HP) production and a 1.5-fold increase in lycopene production. Collectively, this work provides a simple and effective strategy for tunable gene regulation in V. natriegens and expands its potential as a versatile platform for the production of value-added chemicals.