Marker-Free CRISPR/Cas9 RNP Transformation for Gene Disruption in Aspergillus fumigatus

Area of Science:

• Molecular Biology
• Mycology
• Genetic Engineering

Background:

• Filamentous fungal pathogens pose significant human health risks.
• CRISPR-based technologies offer advanced genetic engineering capabilities.
• Aspergillus fumigatus is a prevalent human fungal pathogen requiring improved study methods.

Purpose of the Study:

• To present a novel protocol for CRISPR-Cas9 mediated genetic engineering.
• To enable selection marker-free transformation in filamentous fungi.
• To facilitate adaptable genetic manipulation across fungal species.

Main Methods:

• Utilized CRISPR-Cas9 system for targeted gene editing.
• Developed a selection marker-free transformation protocol.
• Optimized the protocol for filamentous fungi, including Aspergillus fumigatus.

Main Results:

• Successfully achieved marker-free CRISPR-Cas9 transformation in filamentous fungi.
• Demonstrated the protocol's adaptability to multiple strains and species.
• Established a more efficient genetic engineering method for fungal pathogens.

Conclusions:

• The described protocol significantly advances genetic engineering in filamentous fungi.
• This method provides a versatile tool for studying fungal pathogens like Aspergillus fumigatus.
• Selection marker-free CRISPR-Cas9 transformation broadens the scope of fungal genetic research.