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

Streptomyces fradiae 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 S. fradiae. 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 S. fradiae Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor P tipA were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene neoI 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 nagB, replacement of P frr with P ermE *, and integration of exogenous vgbS, with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in S. fradiae. Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in S. fradiae, paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of Streptomyces.