Optimizing genome editing efficiency in via a CRISPR/Cas9n-mediated editing system.

is an important bioresource to produce various antibacterial natural products, however, the time-consuming and labor-intensive genome editing toolkits hindered the construction and application of engineered strains, and this study aimed to establish an efficient CRISPR/Cas9n genome editing system in . Initially, the CRISPR/Cas9-mediated editing tool was employed to replace those awkward genome editing tools that relied on homologous recombination, while the off-target Cas9 exhibited high toxicity to Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor P were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene and long fragment sequence (13.3 kb) were achieved with efficiencies of 77.8% and 44%, respectively. Additionally, the established tool was applied to facilitate the rapid deletion of , replacement of P with P*, and integration of exogenous , with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in . Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in , paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of .IMPORTANCEThis study describes the development and application of a genome editing system mediated by CRISPR/Cas9n in for the first time, which overcomes the challenges associated with genome editing caused by high GC content (74.5%) coupling with complex genome structure, and reduces the negative impact of "off-target effect." Our work not only provides a facile editing tool for constructing strains of high-yield neomycin but also offers the technical guidance for the design of a CRISPR/Cas9n mediated genome editing tool in those creatures with high GC content genomes.