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Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum
Kévin Ravenel1, Wilfried Poirier1, Bienvenue Razafimandimby1
1IRF (Infections Respiratoires Fongiques), Univ Angers, Univ Brest, SFR ICAT 4208, Angers, France.
Abstract:
Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.
Insights
CRISPR-Cas9 technology was optimized for Scedosporium species, enabling efficient gene disruption. This advancement facilitates functional genomic studies in these opportunistic fungal pathogens.
Area of Science:
- Mycology
- Molecular Biology
- Genetic Engineering
Background:
- Scedosporium species are opportunistic fungal pathogens causing diverse human infections.
- Limited genetic tools hinder understanding of Scedosporium pathogenic mechanisms.
- Functional genomic studies in Scedosporium are complicated by high non-homologous end joining rates.
Purpose of the Study:
- To optimize CRISPR-Cas9 technology for gene editing in Scedosporium species.
- To develop efficient methods for gene disruption in both wild-type and mutant Scedosporium strains.
- To facilitate future functional genomic and complementation studies in Scedosporium.
Main Methods:
- Utilized in vitro assembled Cas9 ribonucleoprotein (RNP) complexes for CRISPR-Cas9 gene editing.
- Developed a dual RNA-guided Cas9 system for targeted gene cleavage and disruption via homologous recombination in a KU70 deletion mutant.
- Employed two Cas9 RNP complexes for efficient gene deletion in the wild-type strain without prior KU70 disruption.
Main Results:
- Successfully disrupted four dioxygenase genes in the ΔKU70 Scedosporium mutant using the dual Cas9 RNP approach with a hygromycin resistance cassette.
- Achieved efficient deletion of a dioxygenase gene in the wild-type strain using only two Cas9 RNP complexes, with up to 20% efficiency.
- Demonstrated that the optimized CRISPR-Cas9 methods overcome limitations associated with non-homologous recombination and limited selection markers.
Conclusions:
- Optimized CRISPR-Cas9 RNP technology provides a powerful and efficient tool for genetic manipulation in Scedosporium species.
- The developed methods enable targeted gene disruption, paving the way for advanced functional genomic analyses.
- These advancements are crucial for understanding the pathogenicity of Scedosporium and developing targeted interventions.
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