Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.

IF 2.9 3区 生物学 Q2 MYCOLOGY
Kévin Ravenel, Wilfried Poirier, Bienvenue Razafimandimby, Jean-Philippe Bouchara, Amandine Gastebois, Sandrine Giraud
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引用次数: 0

Abstract

Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.

尖孢梭孢基因组编辑CRISPR-Cas9技术的优化
隐孢子菌是一种机会致病菌,可引起各种各样的人类感染。迄今为止,关于这些真菌的致病机制的信息有限,部分原因是可用的遗传工具数量有限。本研究利用体外组装的Cas9核糖核蛋白(RNP)复合物对丝状真菌的功能基因组研究进行了优化,为丝状真菌的功能基因组研究提供了有希望的结果。在这些真菌中,由于非同源重组的高频率,在野生型菌株中进行功能基因组研究特别复杂。编码非同源末端连接系统组分之一的KU70基因需要事先被破坏,这就需要使用第一选择标记。使用双rna引导的Cas9复合物在两端切割靶基因,然后与含有湿霉素抗性基因的修复模板重组,允许在ΔKU70突变体中破坏靶基因。成功地破坏了4个编码双加氧酶的基因,并在5 μg的HygR修复盒中观察到最佳效率。或者,在野生型菌株中,仅使用两个Cas9 RNP复合物就足以实现有效的删除方法;在获得的菌落中,有20%的双加氧酶基因被成功地删除。这些最后的实验条件为多基因缺失和互补实验铺平了道路,这是我们的第一个程序无法达到的,因为只有两个选择标记可用于Scedosporium物种。
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来源期刊
Mycopathologia
Mycopathologia 生物-真菌学
CiteScore
6.80
自引率
3.60%
发文量
76
审稿时长
3 months
期刊介绍: Mycopathologia is an official journal of the International Union of Microbiological Societies (IUMS). Mycopathologia was founded in 1938 with the mission to ‘diffuse the understanding of fungal diseases in man and animals among mycologists’. Many of the milestones discoveries in the field of medical mycology have been communicated through the pages of this journal. Mycopathologia covers a diverse, interdisciplinary range of topics that is unique in breadth and depth. The journal publishes peer-reviewed, original articles highlighting important developments concerning medically important fungi and fungal diseases. The journal highlights important developments in fungal systematics and taxonomy, laboratory diagnosis of fungal infections, antifungal drugs, clinical presentation and treatment, and epidemiology of fungal diseases globally. Timely opinion articles, mini-reviews, and other communications are usually invited at the discretion of the editorial board. Unique case reports highlighting unprecedented progress in the diagnosis and treatment of fungal infections, are published in every issue of the journal. MycopathologiaIMAGE is another regular feature for a brief clinical report of potential interest to a mixed audience of physicians and laboratory scientists. MycopathologiaGENOME is designed for the rapid publication of new genomes of human and animal pathogenic fungi using a checklist-based, standardized format.
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