| Summary: | <i>Trichophyton rubrum</i> is the most prevalent causative agent responsible for 80–90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanisms of disease pathogenesis in <i>T. rubrum</i>. Here, a dual-plasmid-based CRISPR/Cas9 strategy to edit pH regulatory transcription factor, <i>pacC</i>, of a clinical isolate of <i>T. rubrum</i> by non-homologous end joining (NHEJ) repair is presented. A cas9–eGFP fusion that aids pre-screening of primary transformants through detection of GFP fluorescence is expressed from one plasmid while target-specific sgRNA from the other brings about mutagenesis of <i>pacC</i> with an overall efficiency of 33.8–37.3%. The mutants had reduced transcript levels of <i>pacC</i> at both acidic and alkaline pH with several morphological abnormalities. We believe this dual-plasmid-based CRISPR/Cas9 strategy will aid functional genomics studies, especially in non-lab-adapted clinical strains of <i>T. rubrum</i>.
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