RAGATH-Associated DNA Nuclease Assisted DNA Insertion in Corynebacterium glutamicum

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Molecular genetics

Background:

• *Corynebacterium glutamicum* is a crucial microbial host for industrial production of feed and food ingredients.
• Existing long DNA fragment insertion technologies, like Cas9-RecET, are limited to 7.5 kb insertions.
• Advanced genome editing tools are needed to expand the engineering capacity of *C. glutamicum*.

Purpose of the Study:

• To develop an optimized genome editing system for larger DNA fragment insertion in *C. glutamicum*.
• To overcome the limitations of existing Cas9 and Cpf1 systems regarding DNA insertion size.
• To identify novel nucleases capable of efficient, large-scale DNA integration.

Main Methods:

• Systematic evaluation of Cas9, gRNA, and recombinase expression with 17 promoter/plasmid combinations.
• Implementation and testing of the Cpf1 system for DNA insertion.
• Screening of six smaller RAGATH-associated DNA nucleases for cleavage activity in *C. glutamicum*.

Main Results:

• An optimized genome editing vector enabled 8.0 kb DNA insertions in *C. glutamicum* using Cas9.
• The Cpf1 system also achieved 8.0 kb insertions, but plasmid capacity limited larger fragments.
• Two RAGATH-associated DNA nucleases demonstrated superior editing efficiency, enabling integration of up to 11.3 kb DNA fragments.

Conclusions:

• RAGATH-associated DNA nucleases overcome previous size limitations for long DNA fragment insertion in *C. glutamicum*.
• These novel systems significantly advance metabolic engineering and fundamental research capabilities.
• The developed tools expand the potential of *C. glutamicum* as a microbial chassis.