A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.
A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.
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DOI：
10.1371/journal.pone.0133085
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Mortensen UH
中科院分区：
文献类型：
--
作者：
Nødvig CS;Nielsen JB;Kogle ME;Mortensen UH
完全测序的真菌基因组的数量正在迅速增加。由于大多数丝状真菌的遗传工具开发不足，目前很难利用遗传工程来了解这些真菌的生物学，并在工业上充分利用它们。因此，需要开发可用于遗传操作非模式丝状真菌的通用方法。为了促进这一点，我们开发了一种基于CRISPR-Cas9的系统，适用于丝状真菌。该系统是简单和通用的，因为RNA引导的诱变可以通过用单个质粒转化靶真菌来实现。该系统目前包含四种CRISPR-Cas9载体，这些载体配备了常用的真菌标记，允许在广泛的真菌中进行选择。此外，我们已经开发了一个脚本，允许识别原型间隔区，在多个物种中的目标基因同源物，以促进在不同的丝状真菌中引入常见的突变。利用这些工具，我们已经在六个物种中进行了RNA引导的诱变，其中一个物种以前没有进行过基因工程。此外，对于野生型棘孢曲霉菌株，我们已经使用我们的CRISPR Cas9系统来产生含有AACU_pyrG标记的菌株，并证明所得菌株可用于迭代基因靶向。
The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting.