RAGATH-Associated DNA Nuclease Assisted DNA Insertion in <i>Corynebacterium glutamicum</i>

<i>Corynebacterium glutamicum</i> serves as a key microbial chassis for the industrial production of feed and food ingredients. While long DNA fragment insertion technologies have advanced strain engineering capabilities, previous approaches such as utilizing a chromosome-integrated Cas9-RecET system were constrained by a maximum insertion fragment size of 7.5 kb. Through systematic evaluation of Cas9, gRNA, and recombinase expression driven by five distinct promoters and their implementation on 1 or 2 plasmids with compatible replicons (resulting in a total of 17 combinations), we developed an optimized genome editing vector capable of inserting DNA fragments of up to 8.0 kb in <i>C. glutamicum</i>. Parallel implementation of the Cpf1 system also successfully achieved 8.0 kb of DNA insertions. However, the construction of plasmids carrying insertion sequences larger than 8.0 kb was hindered by the plasmid vector capacity. To address this limitation, we screened six smaller RAGATH-associated DNA nucleases, ultimately identifying two with high cleavage activity in <i>C. glutamicum</i>. These nucleases demonstrated superior editing efficiencies compared to both Cas9 and Cpf1, enabling the integration of DNA fragments up to 11.3 kb─surpassing previously reported size limitations for <i>C. glutamicum</i>. These RAGATH-associated DNA nuclease-based systems effectively overcome the previous size constraints for long fragment insertions, thereby advancing metabolic engineering and fundamental research applications.