[An efficient marker-free genome editing method for Aspergillus niger].

Area of Science:

• Molecular Biology
• Microbial Biotechnology
• Genome Engineering

Context:

• Aspergillus niger is a key industrial microorganism for producing enzymes and organic acids.
• Improving A. niger strains through genome modification is crucial for enhanced industrial output.
• Existing CRISPR/Cas9 methods for A. niger often require selection markers or suffer from low efficiency.

Purpose:

• To develop a highly efficient, marker-free genome editing system for Aspergillus niger.
• To overcome limitations of current CRISPR/Cas9 applications in industrial A. niger strains.
• To facilitate gene function studies and the construction of improved microbial cell factories.

Summary:

• A novel CRISPR/Cas9 system was established using a co-expression plasmid with an AMA1 replication region and the 5S rRNA promoter for improved sgRNA expression.
• A non-homologous end-joining deficient strain (kusA knockout) was created to enhance editing efficiency.
• The system achieves 100% gene editing efficiency using marker-free donor DNA and a 20 bp homologous arm, with efficient plasmid clearance via AMA1 instability.

Impact:

• Enables efficient and simplified genome modification of industrial Aspergillus niger strains.
• Facilitates rapid investigation of gene functions in A. niger.
• Accelerates the development of advanced microbial cell factories for biotechnological applications.