Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatusCitation formats

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Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus. / Van Rhijn, Norman; Furukawa, Takanori; Zhao, Can; Mccann, Bethany; Bignell, Elaine; Bromley, Michael.

In: Fungal Genetics and Biology, 20.10.2020.

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@article{a77e7ddac88642d6be389f1f33e6f157,
title = "Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus",
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 repairtemplate used during this transformation can be obtained with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.",
author = "{Van Rhijn}, Norman and Takanori Furukawa and Can Zhao and Bethany Mccann and Elaine Bignell and Michael Bromley",
year = "2020",
month = oct,
day = "20",
language = "English",
journal = "Fungal Genetics and Biology",
issn = "1087-1845",
publisher = "Elsevier BV",

}

RIS

TY - JOUR

T1 - Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus

AU - Van Rhijn, Norman

AU - Furukawa, Takanori

AU - Zhao, Can

AU - Mccann, Bethany

AU - Bignell, Elaine

AU - Bromley, Michael

PY - 2020/10/20

Y1 - 2020/10/20

N2 - 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 repairtemplate used during this transformation can be obtained with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.

AB - 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 repairtemplate used during this transformation can be obtained with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.

M3 - Article

JO - Fungal Genetics and Biology

JF - Fungal Genetics and Biology

SN - 1087-1845

ER -