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CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis
Published on: June 9, 2020
Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus
Norman van Rhijn1, Takanori Furukawa1, Can Zhao1
1Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health, University of Manchester, CTF Building, 46 Grafton Street, Manchester M13 9NT, UK; Lydia Becker Institute of Immunology and Inflammation, Manchester Collaborative Centre for Inflammation Research, Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.
Abstract:
Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.
Insights
This study introduces a faster, marker-free CRISPR-Cas9 gene editing method for Aspergillus fumigatus. This approach simplifies genetic manipulation, aiding research into fungal infections.
Area of Science:
- Medical Mycology
- Molecular Biology
- Genetics
Background:
- Aspergillus fumigatus causes over 300,000 life-threatening infections yearly.
- Understanding A. fumigatus pathogenesis and disease progression is limited.
- Rapid and versatile gene editing tools are crucial for A. fumigatus research.
Purpose of the Study:
- To develop a faster, selection-free genome editing methodology for A. fumigatus.
- To overcome limitations of current multi-step cloning and selection marker procedures.
- To enable efficient protein tagging and site-directed mutagenesis.
Main Methods:
- Utilized an in vitro CRISPR-Cas9 assembly methodology.
- Employed a selection-free genome editing approach.
- Designed repair templates with 50 bp micro-homology arms, generated via single PCR or synthesized oligonucleotides.
Main Results:
- Successfully performed selection-free genome editing in A. fumigatus.
- Enabled epitope tagging of proteins and site-directed mutagenesis.
- Significantly decreased the time required for complex construct synthesis.
Conclusions:
- The in vitro CRISPR-Cas9 assembly method offers a rapid and versatile tool for A. fumigatus gene editing.
- This selection-free approach simplifies genetic manipulation, reducing host burden and unintended modifications.
- The methodology accelerates research into A. fumigatus pathogenesis and antifungal drug development.
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