Statitstical Analysis and supplemental material Gonzalez et al 2025

BackgroundHistoplasmosis is a systemic and endemic mycosis caused by the dimorphic fungus belonging to the genus Histoplasma. The survival and intracellular replication of this pathogen rely on the acquisition of micronutrients like zinc, which functions as essential cofactors and cellular signalers. This study aims to generate a Histoplasma mutant for the gene encoding the zinc transporter protein ZRT2 using the CRISPR-Cas9 genetic editing system.MethodologyA targeting plasmid vector, expressing Cas9 and one single guide RNA using the promoter of CBP1 (calcium-binding protein), was commercially synthesized based on the genome sequence of the Histoplasma, and this plasmid was introduced into Histoplasma via Agrobacterium tumefaciens gene transfer. Additionally, expression of the ZRT2 gene was evaluated under different zinc-limitation conditions.ResultsWe successfully obtained a disrupted ZRT2 mutant, which showed a significant reduction in the gene expression; however, the mutant was lost at early time points in low-passage-number cultures, as confirmed by whole-genome sequencing.ConclusionsThe above results suggest that ZRT2 could be involved in scavenging and uptake of zinc and confirm that the editing system used to transform fungal cells needs refinement.