Editing gliA, gliP and gliZ of Aspergillus fumigatus Using CRISPR/Cas System Renders Fungus Incapable to Produce Gliotoxin

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.