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首页> 外文期刊>Current Protocols in Microbiology >Gene Editing in Dimorphic Fungi Using CRISPR/Cas9
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Gene Editing in Dimorphic Fungi Using CRISPR/Cas9

机译：使用CRISPR / Cas9基因编辑双晶的真菌

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摘要

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 particularas 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.
机译：二态的真菌属芽生菌,组织胞浆菌属、球孢子菌属和Paracoccidioides是重要的人类病原体影响人类全世界许多国家的卫生。理解这些真菌的生物学重要的有效的发展治疗和疫苗。研究这些至关重要的工具生物。方法采用RNA检测引导DNA核酸酶,如定期聚集空间短回文的重复(CRISPR) / CRISPR相关联核酸酶9 (Cas9),人气暴涨。在这里,我们提供的详细描述应用CRISPR / Cas9技术所涉及的步骤皮炎芽生菌与二态的真菌模型在particularas真菌病原体。讨论单的设计与施工指导RNA和Cas9表达目标向量(包括多路复用向量)以及引入这些质粒成芽生菌利用农杆菌介导转化。最后,我们的结果预期的编辑效率和基因检测类型的基因改变。

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