Establishing a one-step marker-free CRISPR/Cas9 system for industrial Aspergillus niger using counter-selectable marker Ang-ace2

To develop a one-step, marker-free CRISPR/Cas9 system for highly efficient genome editing in industrial Aspergillus niger, with a short genetic operation cycle.Firstly, evaluation of different promoters for sgRNA expression revealed tRNAGly15 as the most efficient, achieving a remarkable 100% gene editing efficiency. Furthermore, a counter-selectable marker, Ang-ace2, was identified for A. niger. Subsequently, a CRISPR/Cas9 plasmid was developed, utilizing a truncated AMA1 element and the Ang-ace2 conditional expression cassette driven by a Tet-on promoter. In the presence of doxycycline, the plasmid demonstrated a 33% loss efficiency in the progeny of A. niger spores after a single generation, resulting in a shortened genetic operation cycle of 16 days for CRISPR/Cas9.The one-step marker-free CRISPR/Cas9 system was successfully developed in industrial A. niger, allowing for efficient gene editing while simultaneously reducing the editing time.