Efficient Multiplex Gene Repression by CRISPR-dCpf1 in Corynebacterium glutamicum.

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Metabolic engineering

Background:

• Corynebacterium glutamicum is a key industrial microorganism for bioproduct synthesis.
• Efficient multiplex gene regulation is essential for optimizing metabolic pathways.
• Limited tools exist for simultaneous control of multiple genes in C. glutamicum.

Purpose of the Study:

• To develop a CRISPR-dCpf1-based system for multiplex gene repression in C. glutamicum.
• To demonstrate the system's efficacy in enhancing lysine biosynthesis.

Main Methods:

• Engineered a catalytically inactive Cas12a (dCpf1) protein.
• Designed a single CRISPR RNA (crRNA) array for multiplex targeting.
• Applied the system to repress four key genes in the lysine biosynthesis pathway (gltA, pck, pgi, hom).

Main Results:

• Successfully repressed two fluorescent reporter genes simultaneously.
• Achieved over 4.0-fold increase in lysine titer and yield.
• Demonstrated over 90% transcriptional repression of four endogenous target genes using quantitative PCR.

Conclusions:

• The CRISPR-dCpf1 system provides a simple and effective method for multiplex gene repression in C. glutamicum.
• This technology holds significant potential for metabolic engineering applications in producing valuable chemicals and fuels.