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Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

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Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers.

Lukas Birštonas¹, Alex Dallemulle¹, Manuel S López-Berges¹

¹Institute of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria.

|April 9, 2020

Summary

This summary is machine-generated.

Researchers developed a new genetic toolbox for fungi using endogenous selectable markers. This innovation enables precise DNA insertions without foreign markers, advancing fungal genetic engineering and research.

Keywords:

endogenous selectable markers genetic engineering pyrimidine salvage pathway targeted genomic integrations

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Area of Science:

Mycology
Molecular Biology
Genetic Engineering

Background:

Selectable markers are crucial for genetic engineering but are limited in number and variety.
Current methods often rely on antibiotic resistance genes, necessitating new, minimally invasive tools.
Sophisticated genetic manipulations require a broader range of selectable markers.

Purpose of the Study:

To characterize three endogenous genes in *Aspergillus fumigatus* as selectable markers for targeted genomic insertions.
To assess the impact of these gene knockouts on fungal growth, stress resistance, and virulence.
To validate the utility of these markers in other fungal species like *Penicillium chrysogenum* and *Fusarium oxysporum*.

Main Methods:

Identified and characterized three endogenous genes involved in pyrimidine metabolism in *A. fumigatus*.
Utilized resistance to 5-fluorocytosine and 5-fluorouracil as selection criteria for successful DNA insertion.
Demonstrated individual and sequential marker use for integrating reporter genes and fluorescent proteins.

Main Results:

DOI integration, leading to marker gene inactivation, conferred resistance to 5-fluorocytosine and 5-fluorouracil.
Inactivated marker genes had no negative impact on nutrient requirements, stress resistance, or virulence.
Successfully demonstrated sequential multicolor localization microscopy using three fluorescent protein markers in a single strain.
Validated marker applicability in *Penicillium chrysogenum* and *Fusarium oxysporum*.

Conclusions:

A novel toolbox of endogenous selectable markers for targeted genomic insertions in fungi was discovered.
This technology facilitates multiple, site-specific DNA insertions without the need for exogenous markers.
The findings significantly advance genetic and metabolic engineering capabilities in fungal species.