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CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species

Remi Hatinguais1, Ian Leaves1, Gordon D Brown1

  • 1Medical Research Council Centre for Medical Mycology, University of Exeter , Exeter, United Kingdom.

Microbiology Spectrum
|September 14, 2023
PubMed
Abstract

Insights

Researchers developed a new CRISPR/Cas9 gene editing tool for Sporothrix fungi, enabling targeted gene deletion. This advances understanding of sporotrichosis virulence factors and aids in developing new treatments.

Area of Science:

  • Mycology and Infectious Diseases
  • Molecular Biology and Genetics

Background:

  • Sporothrix brasiliensis causes sporotrichosis, an emerging zoonotic disease spreading rapidly in Latin America.
  • Limited molecular tools hinder the study of Sporothrix virulence factors and disease mechanisms.
  • Understanding fungal pathogenesis is crucial for developing effective diagnostics and therapies.

Purpose of the Study:

  • To establish a robust method for genetic manipulation in Sporothrix species.
  • To enable targeted gene deletion for studying virulence factors.
  • To overcome limitations in reverse genetics for Sporothrix research.

Main Methods:

  • Developed a PEG-mediated protoplast transformation protocol for heterologous gene expression.
  • Utilized CRISPR/Cas9 gene editing combined with the transformation protocol.
  • Deleted the putative DHN-melanin synthase gene (pks1) and the KU80 homolog to enhance homologous recombination.

Main Results:

  • Successfully demonstrated heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis.
  • Enabled targeted deletion of the pks1 gene, a proposed virulence factor.
  • Deletion of KU80 enhanced homologous-directed repair, improving targeted gene deletion efficiency with CRISPR/Cas9.

Conclusions:

  • The developed CRISPR/Cas9-based transformation protocol is an efficient tool for targeted gene manipulation in Sporothrix species.
  • This advancement addresses the scarcity of reliable genetic engineering tools for Sporothrix.
  • Facilitates the study of virulence determinants and their roles in sporotrichosis pathogenesis.
Keywords:
CRISPR/Cas9DHN-melaninSporothrixku80pks1

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