Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in <i>Pneumocystis murina</i>

<i>Pneumocystis</i> species are obligate fungal pathogens that cause severe pneumonia, particularly in immunocompromised individuals. The absence of robust genetic manipulation tools has impeded our mechanistic understanding of <i>Pneumocystis</i> biology and the development of novel therapeutic strategies. Herein, we describe a novel method for the stable transformation and CRISPR/Cas9-mediated genetic editing of <i>Pneumocystis murina</i> utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to <i>Pneumocystis</i>, we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful <i>in vitro</i> transformation and subsequent expression of <i>mNeonGreen</i> and <i>Dhps^ARS</i> in <i>P. murina</i> organisms. Subsequently, we established stable <i>in vivo</i> expression of <i>mNeonGreen</i> in mice infected with transformed <i>P. murina</i> for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the <i>P. murina Dhps</i> gene, confirming DNA cleavage efficiency <i>in vitro</i>. Ultimately, we achieved successful <i>in vivo</i> CRISPR/Cas9-mediated homologous recombination, precisely introducing a <i>Dhps^ARS</i> mutation into the <i>P. murina</i> genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in <i>Pneumocystis</i>, thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.IMPORTANCE<i>Pneumocystis</i> species are obligate fungal pathogens and major causes of pneumonia in immunocompromised individuals. However, their strict dependence on the mammalian lung environment has precluded the development of genetic manipulation systems, limiting our ability to interrogate gene function, study antifungal resistance mechanisms, or validate therapeutic targets. Here, we report the first successful approach for stable transformation and CRISPR/Cas9-based genome editing of <i>Pneumocystis murina</i>, achieved through <i>in vivo</i> delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the <i>dhps</i> locus via homology-directed repair. This modular, scalable platform overcomes a long-standing barrier in the field and establishes a foundation for functional genomics in <i>Pneumocystis</i> and other obligate, host-adapted microbes.