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Gene Editing in Dimorphic Fungi Using CRISPR/Cas9
Gregory C Kujoth1, Thomas D Sullivan1, Bruce S Klein1,2
1Department of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin.
Abstract:
Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks.
Insights
This study details using CRISPR/Cas9 gene editing in dimorphic fungi, specifically Blastomyces dermatitidis. This powerful technique enables precise genetic modifications for better understanding and treatment of fungal pathogens.
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.
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