Related Experiment Video
Updated: May 16, 2025
CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans
Published on: November 14, 2018
Editing gliA, gliP and gliZ of Aspergillus fumigatus Using CRISPR/Cas System Renders Fungus Incapable to Produce
Rida Zainab1, Arsh Mukhtar1, Zakia Saleem1
1Department of Animal Breeding and Genetics (Section Genetics), University of Veterinary and Animal Sciences, Lahore, Pakistan.
Abstract:
Aspergillus fumigatus is a saprophytic fungus that causes respiratory infections in human, animals, and birds. This fungus produces gliotoxin which is a secondary metabolite that triggers pathogenicity. Gliotoxin is encoded by a 13-gene cluster including gliA, gliP and gliZ. The purpose of this study was to determine whether the fungus produces gliotoxin after these genes are edited using CRISPR/Cas system. For this, crRNAs for gliA, gliP and gliZ were designed using EuPaGDT, while tracrRNA and Cas9 protein were purchased ready-made. These crRNAs were individually annealed with the tracrRNA to make three gRNAs which were then individually combined with the Cas9 to make three ribonucleoprotein (RNP) complexes. A. fumigatus protoplasts were enzymatically generated and transfected with each of the RNP complexes (group 1) in PEGylated conditions. Non-treated protoplasts were simultaneously run as control (group 2). Transfected protoplasts showed reduced growth on SDA plates as compared to their control. Gliotoxin extraction through thin-layer chromatography was carried out for both the groups which showed the absence of gliotoxin in group 1. Sequencing results confirmed the indels in target genes which shows that the CRISPR/Cas9 system effectively targeted A. fumigatus' gliotoxin-related genes that rendered fungus incapable to produce gliotoxin. This work may pave the way to develop effective strategies to control the infections caused by A. fumigatus.
Insights
CRISPR/Cas9 gene editing successfully disabled gliotoxin production in Aspergillus fumigatus. This breakthrough offers potential new strategies for controlling fungal infections by targeting pathogenicity factors.
Area of Science:
- Medical Mycology
- Molecular Biology
- Genetic Engineering
Background:
- Aspergillus fumigatus causes respiratory infections in humans and animals.
- Pathogenicity is linked to the secondary metabolite gliotoxin, encoded by a 13-gene cluster.
- Targeting gliotoxin production could mitigate A. fumigatus virulence.
Purpose of the Study:
- To investigate the efficacy of CRISPR/Cas9 gene editing in disrupting gliotoxin biosynthesis in A. fumigatus.
- To determine if editing gliA, gliP, and gliZ genes prevents gliotoxin production.
Main Methods:
- CRISPR/Cas9 ribonucleoprotein (RNP) complexes were designed targeting gliA, gliP, and gliZ genes.
- A. fumigatus protoplasts were transfected with RNP complexes.
- Gliotoxin production was assessed via thin-layer chromatography and gene sequencing.
Main Results:
- Transfected protoplasts exhibited reduced growth compared to controls.
- Gliotoxin was undetectable in protoplasts treated with CRISPR/Cas9 RNP complexes.
- Sequencing confirmed targeted gene edits (indels) in gliA, gliP, and gliZ.
Conclusions:
- CRISPR/Cas9 effectively targeted and disrupted gliotoxin-related genes in A. fumigatus.
- Gene editing rendered the fungus incapable of producing gliotoxin.
- This study provides a foundation for developing novel anti-fungal strategies targeting gliotoxin.
