A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Microbial Strain Development

Background:

• CRISPR interference (CRISPRi) accelerates target identification in platform strain development.
• Existing CRISPRi systems may lack simplicity or dual-inducibility for industrial applications.
• Corynebacterium glutamicum is a key industrial microorganism for biotechnological production.

Purpose of the Study:

• To engineer a simple, reliable, and dual-inducible CRISPRi system for Corynebacterium glutamicum.
• To enable precise temporal and spatial control over gene expression in C. glutamicum.
• To facilitate rapid evaluation of gene function and pathway engineering strategies.

Main Methods:

• Developed a single plasmid system combining anhydro-tetracycline-inducible dCas9 and IPTG-inducible single guide RNA (sgRNA) expression.
• Demonstrated functionality by targeting the l-arginine biosynthesis pathway, specifically the argininosuccinate lyase gene (argH).
• Utilized multiplex targeting by concatenating multiple sgRNA sequences on one plasmid to down-regulate multiple genes simultaneously (e.g., argH and pgi).

Main Results:

• Successfully established a dual-inducible CRISPRi system in C. glutamicum.
• Demonstrated precise down-regulation of argH, leading to a metabolic shift from l-arginine to citrulline production.
• Showcased multiplex gene targeting capability by simultaneously down-regulating argH and phosphoglucose isomerase (pgi).

Conclusions:

• The developed dual-inducible CRISPRi system is a powerful and versatile tool for pathway engineering in C. glutamicum.
• This system significantly enhances the efficiency of target gene evaluation compared to traditional knockout methods.
• It offers a promising approach for accelerating microbial strain development and optimizing biotechnological processes.