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

Rhijn, Norman van; Furukawa, Tatsuhiko; Zhao, Can; McCann, Bethany L.; Bignell, Elaine; Bromley, Michael

doi:10.1016/j.fgb.2020.103479

Cited by 38 publications

(54 citation statements)

References 41 publications

(38 reference statements)

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“…Despite the recent development of marker-free technologies and marker-recycling strategies [ 32 , 33 , 34 ], dominant selectable markers are still widely used in molecular genetics, particularly for in vitro manipulation [ 7 , 35 ]. Whilst the absence of marker cassettes, in the form of exogenous DNA, presents considerable advantages [ 36 ], the possibility to verify the purity of the mutant strains using selective conditions at any time and the transformation efficiency reached when employing dominant markers represent two significant benefits of opting for the transformation approach.…”

Section: Discussionmentioning

confidence: 99%

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

Baldin

Kühbacher

Merschak

et al. 2021

JoF

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The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

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Section: Discussionmentioning

confidence: 99%

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

Baldin

Kühbacher

Merschak

et al. 2021

JoF

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“… 2016 ) and A. fumigatus (van Rhijn et al . 2020 ). Being able to selectively and efficiently manipulate specific loci, will not only enable expansion of existing libraries, but also facilitate creation of libraries in differing genetic backgrounds with varying environmental origins.…”

Section: Discussionmentioning

confidence: 99%

Hsp90 interaction networks in fungi—tools and techniques

Crunden

Diezmann

2021

FEMS Yeast Research

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Heat-shock protein 90 (Hsp90) is a central regulator of cellular proteostasis. It stabilizes numerous proteins that are involved in fundamental processes of life, including cell growth, cell-cycle progression and the environmental response. In addition to stabilizing proteins, Hsp90 governs gene expression and controls the release of cryptic genetic variation. Given its central role in evolution and development, it is important to identify proteins and genes that interact with Hsp90. This requires sophisticated genetic and biochemical tools, including extensive mutant collections, suitable epitope tags, proteomics approaches and Hsp90-specific pharmacological inhibitors for chemogenomic screens. These usually only exist in model organisms, such as the yeast Saccharomyces cerevisiae . Yet, the importance of other fungal species, such as Candida albicans and Cryptococcus neoformans , as serious human pathogens accelerated the development of genetic tools to study their virulence and stress response pathways. These tools can also be exploited to map Hsp90 interaction networks. Here, we review tools and techniques for Hsp90 network mapping available in different fungi and provide a summary of existing mapping efforts. Mapping Hsp90 networks in fungal species spanning >500 million years of evolution provides a unique vantage point, allowing tracking of the evolutionary history of eukaryotic Hsp90 networks.

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“…The CYP51A2 gene was amplified from A. fumigatiaffinis genomic DNA using primers cyp51a2_Fw and cyp51a2_Rv ( Table S2 ) including 1.5 kb upstream and 500 bp downstream of the predicted open reading frame (ORF), followed by gel purification (NucleoSpin; Macherey-Nagel). CYP51A2 was transformed into a genomic “safe haven” in A. fumigatus A1160p+ using selection-free CRISPR-Cas9-mediated transformation with crRNA atf4_TIRrm_up and aft4_TIRrm_down ( 22 , 23 ). Transformants were purified twice on Sabouraud agar and PCR verified using cyp51a2_chk_fw and aft4_screening_rv.…”

Section: Observationmentioning

confidence: 99%

CYP51 Paralogue Structure Is Associated with Intrinsic Azole Resistance in Fungi

Rhijn

Bromley

Bowyer

2021

mBio

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

Abstract: Highlights In vitro assembled CRISPR/Cas9-gRNA complexes can mediate selection free transformation. Single gRNAs can be used to target mutagenesis effectively. Targeted point mutations can be achieved utilising single strand DNA oligonucleotides as repair template.

Cited by 38 publications

References 41 publications

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

Hsp90 interaction networks in fungi—tools and techniques

CYP51 Paralogue Structure Is Associated with Intrinsic Azole Resistance in Fungi

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