Editing Aspergillus terreus using the CRISPR-Cas9 system

Area of Science:

• Molecular Biology
• Mycology
• Biotechnology

Background:

• CRISPR-Cas9 is a versatile tool for targeted mutagenesis in various organisms.
• Its application in Aspergillus terreus for genome editing remains underexplored, lacking established guidelines.
• Developing efficient genome editing protocols is crucial for functional genomics in A. terreus.

Purpose of the Study:

• To establish and optimize CRISPR-Cas9 genome editing in Aspergillus terreus.
• To demonstrate precise gene disruption, insertion, and deletion using the CRISPR-Cas9 system.
• To provide a robust method for gene manipulation and functional studies in A. terreus.

Main Methods:

• Construction of a modified single-guide RNA (sgRNA)/Cas9 expression plasmid for optimized gRNA expression.
• Co-transformation of sgRNA/Cas9 plasmid with marker-free donor DNA for gene disruption and insertion.
• Co-delivery of two sgRNA/Cas9 plasmids for gene deletion and DNA removal between targeting sites.

Main Results:

• Precise disruption of lovB and lovR genes was achieved.
• Targeted insertion of the lovF gene and iterative gene editing of lovF and lovR were successful.
• Efficient deletion of ku70 and pyrG genes, and removal of DNA between targeting sites in pyrG were accomplished.

Conclusions:

• The CRISPR-Cas9 system is a powerful and precise tool for genome editing in Aspergillus terreus.
• The developed approach offers significant potential for targeted gene manipulation.
• This methodology will greatly contribute to the functional study of genes in A. terreus.