Genome editing using a versatile vector-based CRISPR/Cas9 system in Fusarium species

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• Fusarium species are significant fungal pathogens affecting plants, animals, and humans.
• Certain nonpathogenic Fusarium species show potential as biocontrol agents against plant pathogens.

Purpose of the Study:

• To develop an efficient genome editing technology for Fusarium oxysporum f. sp. lycopersici (Fol).
• To create a versatile CRISPR/Cas9 system applicable to various Fusarium species.

Main Methods:

• Development of a vector-based CRISPR/Cas9 system utilizing an endogenous U6 small nuclear RNA promoter for single-guide RNA expression.
• Incorporation of an endogenous H2B nuclear localization signal for Cas9 localization.
• Application of the system for targeted gene knock-out, base editing, and gene tagging in Fusarium species.

Main Results:

• The optimized CRISPR/Cas9 system achieved efficient targeted gene knock-out, including in accessory chromosomal regions of Fol.
• Demonstrated successful single crossover-mediated targeted base editing and endogenous gene tagging.
• The system proved effective for genome editing in F. oxysporum f. sp. spinaciae and F. commune without modifications.

Conclusions:

• The developed CRISPR/Cas9 vector-based system provides an efficient method for genome editing in Fusarium species.
• This technology has broad potential applications for research across a wide range of Fusarium species, including pathogenic and nonpathogenic strains.