Simultaneous multigene integration in Aspergillus fumigatus using CRISPR/Cas9 and endogenous counter-selectable...

Area of Science:

• Mycology
• Molecular Biology
• Genetic Engineering

Background:

• CRISPR/Cas9 revolutionized genome editing, enabling precise genetic modifications.
• Selectable markers are crucial for identifying successfully edited cells.
• Previous methods for multigene integration in Aspergillus fumigatus were time-consuming due to sequential transformations.

Purpose of the Study:

• To develop a streamlined method for simultaneous multigene integration in Aspergillus fumigatus.
• To leverage CRISPR/Cas9 and endogenous counter-selectable markers for efficient genetic manipulation.

Main Methods:

• Coupled CRISPR/Cas9 with three endogenous counter-selectable markers (azgA, fcyB, cntA) for simultaneous cassette integration.
• Utilized markers' roles in nutrient uptake for triple selective transformation.
• Introduced fluorescent reporters to monitor growth patterns in azole-resistant isolates.

Main Results:

• Successfully achieved simultaneous genomic integration of three expression cassettes in A. fumigatus.
• Demonstrated a triple selective transformation procedure.
• Visualized and monitored growth of engineered fungal isolates with varying antifungal resistance.

Conclusions:

• The developed technology significantly reduces time and labor for genetic manipulation in fungi.
• Enables flexible and efficient introduction of multiple genetic traits in a single step.
• Advances rapid and effective multigene integration for fungal genetic engineering applications.