A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in <i>Acremonium chrysogenum</i>

<i>Acremonium chrysogenum</i> 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 for <i>A. chrysogenum</i> employ heterologous promoters, including <i>Aspergillus nidulans</i> P<i>gpdA</i> or <i>Aspergillus fumigatus</i> AfU6p, 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 <i>A. chrysogenum</i>. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting the <i>sorB</i> gene. 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 in <i>A. chrysogenum</i> to date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at the <i>kusA</i> locus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in <i>A. chrysogenum</i>.