Gene Editing in Dimorphic Fungi Using CRISPR/Cas9.

Area of Science:

• Mycology
• Molecular Biology
• Genetic Engineering

Background:

• Dimorphic fungi like Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are significant human pathogens globally.
• Understanding their biology is crucial for developing new treatments and vaccines.
• Gene editing technologies are vital research tools for these organisms.

Purpose of the Study:

• To provide a detailed protocol for applying CRISPR/Cas9 gene editing technology to dimorphic fungi.
• To use Blastomyces dermatitidis as a model organism for demonstrating these gene-editing techniques.
• To guide researchers in designing and implementing gene-editing strategies for fungal pathogens.

Main Methods:

• Design and construction of single-guide RNA and Cas9-expressing targeting vectors, including multiplexed options.
• Introduction of plasmids into Blastomyces using Agrobacterium-mediated transformation.
• Detailed protocols for vector construction, protospacer selection, and Agrobacterium/Blastomyces preparation.

Main Results:

• Demonstration of CRISPR/Cas9 application in Blastomyces dermatitidis.
• Guidance on expected gene-editing efficiency and the types of genetic alterations achievable.
• Successful implementation of Agrobacterium-mediated transformation for gene targeting.

Conclusions:

• CRISPR/Cas9 technology is a powerful and applicable tool for genetic manipulation in dimorphic fungi.
• This protocol facilitates in-depth research into the biology of significant fungal pathogens.
• The described methods will aid in the development of novel therapeutic strategies against fungal infections.