Presentation Information
[3GET-17]Tunable Transcription-Level CRISPR Interference in Vibrio natriegens Using Engineered Single Guide RNAs
○Mincheol Choi1, Gibyuck Byun1, Giho Kim1, Jina Yang2, Sang Woo Seo1 (1. Seoul National University (Korea), 2. Jeju National University (Korea))
Keywords:
CRISPR interference,synthetic sgRNA,tunable gene regulation,3-hydroxypropionic acid,lycopene,Vibrio natriegens
[Purpose]
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 multi-level transcriptional regulation in V. natriegens.
[Method]
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 and predictable repression behavior across multiple protospacer targets.
[Results]
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.
[Conclusion]
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.
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 multi-level transcriptional regulation in V. natriegens.
[Method]
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 and predictable repression behavior across multiple protospacer targets.
[Results]
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.
[Conclusion]
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.
Comment
To browse or post comments, you must log in.Log in