is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems foremploy heterologous promoters, includingPorAfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload. Here, we developed a simplified and highly efficient CRISPR/Cas9 genome editing system using the endogenous 5S rRNA promoter for sgRNA transcription in. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting thegene. Furthermore, this platform enabled 100% single gene deletion and efficient large-scale chromosomal deletion, up to 66.17 kb within the sorbicillinoid biosynthetic gene cluster, without donor DNA. To our knowledge, this represents the largest chromosomal deletion reported into date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at thelocus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in. Acremonium chrysogenum A. chrysogenum Aspergillus nidulans gpdA Aspergillus fumigatus A. chrysogenum sorB A. chrysogenum kusA A. chrysogenum
