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Title: Gene Editing in Dimorphic Fungi Using CRISPR/Cas9
Abstract

Dimorphic fungi in the generaBlastomyces,Histoplasma,Coccidioides, andParacoccidioidesare 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, withBlastomyces dermatitidisin 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 intoBlastomycesusingAgrobacterium‐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 ofBlastomyces

Support Protocol 2: Preparation of electrocompetentAgrobacterium

Support Protocol 3: Preparation and recovery ofBlastomycesfrozen stocks

 
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NSF-PAR ID:
10238511
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols in Microbiology
Volume:
59
Issue:
1
ISSN:
1934-8525
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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