Statitstical Analysis and supplemental material Gonzalez et al 2025

<b><i>Background</i></b>Histoplasmosis is a systemic and endemic mycosis caused by the dimorphic fungus belonging to the genus <i>Histoplasma</i>. 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 <i>Histoplasma</i> mutant for the gene encoding the zinc transporter protein ZRT2 using the CRISPR-Cas9 genetic editing system.<b><i>Methodology</i></b>A 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 <i>Histoplasma</i>, and this plasmid was introduced into <i>Histoplasma</i> via <i>Agrobacterium tumefaciens</i> gene transfer. Additionally, expression of the <i>ZRT2</i> gene was evaluated under different zinc-limitation conditions.<b><i>Results</i></b>We successfully obtained a disrupted <i>ZRT2</i> 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.<b><i>Conclusions</i></b>The above results suggest that <i>ZRT2</i> could be involved in scavenging and uptake of zinc and confirm that the editing system used to transform fungal cells needs refinement.