Journal Article

A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi

Kogle, Martin E., Mortensen, Uffe H., Nødvig, Christina S., Nielsen, Jakob B.

2015

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Aspergillus / Aspergillus - classification / Aspergillus - genetics / Aspergillus - metabolism / Aspergillus nidulans / Base Sequence / Biology / Biotechnology / Cloning / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / Escherichia coli - genetics / Escherichia coli - metabolism / Fungi / Gene Expression Regulation, Fungal / Gene targeting / Gene Targeting - methods / Genes / Genetic engineering / Genetic Engineering - methods / Genetic Markers / Genetically modified organisms / Genomes / Genomics / Homology / Introduced species / Iterative methods / Metabolites / Molecular Sequence Data / Mutagenesis / Mutation / Plasmids - chemistry / Plasmids - metabolism / Publishing industry / Ribonucleic acid / RNA / RNA, Catalytic - genetics / RNA, Catalytic - metabolism / RNA, Guide, CRISPR-Cas Systems - genetics / RNA, Guide, CRISPR-Cas Systems - metabolism / Streptococcus pyogenes / Target recognition / Transformation, Genetic