Cas9 Ribonucleoproteins (RNPs) for Gene Deletion.

Area of Science:

• Molecular Biology
• Mycology
• Genetics

Background:

• Genetic manipulation of pathogenic fungi like Aspergillus fumigatus is crucial for understanding pathogenicity.
• Traditional methods for gene deletion in filamentous fungi involve complex DNA fragment sub-cloning or fusion PCR.
• CRISPR technology has revolutionized genome editing, offering faster and simpler approaches.

Purpose of the Study:

• To develop a simplified and universal one-step CRISPR-Cas9 genetic tool for filamentous fungi.
• To utilize repair templates with microhomology regions for efficient gene targeting.
• To assess the efficiency and applicability of this system across different fungal genetic backgrounds.

Main Methods:

• A one-step CRISPR-Cas9 gene targeting system was designed.
• Repair templates incorporating microhomology regions were employed.
• The system was tested for its efficiency in gene deletion and its adaptability.

Main Results:

• The developed CRISPR-Cas9 tool demonstrated high homologous recombination efficiency.
• The system proved to be simple and universally applicable across various genetic backgrounds.
• This method significantly accelerates the genome editing process in fungi.

Conclusions:

• The described one-step CRISPR-Cas9 system offers a highly efficient and user-friendly approach for fungal gene editing.
• Microhomology-based repair templates enhance the effectiveness of CRISPR-mediated gene targeting.
• This technology has broad implications for studying gene function in pathogenic fungi and other applications.