{"doi":"10.1128/mbio.01825-25","title":"Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in\n                    <i>Pneumocystis murina</i>","abstract":"<jats:title>ABSTRACT</jats:title>\n                  <jats:sec>\n                    <jats:title/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Pneumocystis murina</jats:italic>\n                      utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      , we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful\n                      <jats:italic toggle=\"yes\">in vitro</jats:italic>\n                      transformation and subsequent expression of\n                      <jats:italic toggle=\"yes\">mNeonGreen</jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">\n                        Dhps\n                        <jats:sup>ARS</jats:sup>\n                      </jats:italic>\n                      in\n                      <jats:italic toggle=\"yes\">P. murina</jats:italic>\n                      organisms. Subsequently, we established stable\n                      <jats:italic toggle=\"yes\">in vivo</jats:italic>\n                      expression of\n                      <jats:italic toggle=\"yes\">mNeonGreen</jats:italic>\n                      in mice infected with transformed\n                      <jats:italic toggle=\"yes\">P. murina</jats:italic>\n                      for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the\n                      <jats:italic toggle=\"yes\">P. murina Dhps</jats:italic>\n                      gene, confirming DNA cleavage efficiency\n                      <jats:italic toggle=\"yes\">in vitro</jats:italic>\n                      . Ultimately, we achieved successful\n                      <jats:italic toggle=\"yes\">in vivo</jats:italic>\n                      CRISPR/Cas9-mediated homologous recombination, precisely introducing a\n                      <jats:italic toggle=\"yes\">\n                        Dhps\n                        <jats:sup>ARS</jats:sup>\n                      </jats:italic>\n                      mutation into the\n                      <jats:italic toggle=\"yes\">P. murina</jats:italic>\n                      genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      , thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.\n                    </jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE</jats:title>\n                      <jats:p>\n                        <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">Pneumocystis murina</jats:italic>\n                        , achieved through\n                        <jats:italic toggle=\"yes\">in vivo</jats:italic>\n                        delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the\n                        <jats:italic toggle=\"yes\">dhps</jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                        and other obligate, host-adapted microbes.\n                      </jats:p>\n                    </jats:sec>\n                  </jats:sec>","journal":"mBio","year":2025,"id":632076,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":538393,"name":"Alan Ashbaugh","orcid":null,"position":1,"is_corresponding":false},{"id":1638316,"name":"Lillian C. Bauer","orcid":null,"position":2,"is_corresponding":false},{"id":1128014,"name":"A. George Smulian","orcid":"0000-0003-3272-0697","position":3,"is_corresponding":false},{"id":691070,"name":"Steven G. Sayson","orcid":"0000-0002-7461-0704","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in\n                    <i>Pneumocystis murina</i>","abstract":"<jats:title>ABSTRACT</jats:title>\n                  <jats:sec>\n                    <jats:title/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Pneumocystis murina</jats:italic>\n                      utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      , we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful\n                      <jats:italic toggle=\"yes\">in vitro</jats:italic>\n                      transformation and subsequent expression of\n                      <jats:italic toggle=\"yes\">mNeonGreen</jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">\n                        Dhps\n                        <jats:sup>ARS</jats:sup>\n                      </jats:italic>\n                      in\n                      <jats:italic toggle=\"yes\">P. murina</jats:italic>\n                      organisms. Subsequently, we established stable\n                      <jats:italic toggle=\"yes\">in vivo</jats:italic>\n                      expression of\n                      <jats:italic toggle=\"yes\">mNeonGreen</jats:italic>\n                      in mice infected with transformed\n                      <jats:italic toggle=\"yes\">P. murina</jats:italic>\n                      for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the\n                      <jats:italic toggle=\"yes\">P. murina Dhps</jats:italic>\n                      gene, confirming DNA cleavage efficiency\n                      <jats:italic toggle=\"yes\">in vitro</jats:italic>\n                      . Ultimately, we achieved successful\n                      <jats:italic toggle=\"yes\">in vivo</jats:italic>\n                      CRISPR/Cas9-mediated homologous recombination, precisely introducing a\n                      <jats:italic toggle=\"yes\">\n                        Dhps\n                        <jats:sup>ARS</jats:sup>\n                      </jats:italic>\n                      mutation into the\n                      <jats:italic toggle=\"yes\">P. murina</jats:italic>\n                      genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in\n                      <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                      , thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.\n                    </jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE</jats:title>\n                      <jats:p>\n                        <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">Pneumocystis murina</jats:italic>\n                        , achieved through\n                        <jats:italic toggle=\"yes\">in vivo</jats:italic>\n                        delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the\n                        <jats:italic toggle=\"yes\">dhps</jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">Pneumocystis</jats:italic>\n                        and other obligate, host-adapted microbes.\n                      </jats:p>\n                    </jats:sec>\n                  </jats:sec>","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"40985725","pmcid":"PMC12607567","openalex_id":"https://openalex.org/W4414425425","authors":[],"funders":[{"funder_name":"National Institute of Allergy and Infectious Diseases","grant_id":"1R61AI187097","title":null},{"funder_name":"NIAID NIH HHS","grant_id":"R61 AI187097","title":null}],"total_grants":2,"fwci":0.0,"citation_percentile":0.28298715,"influential_citations":0,"citation_trend":[],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://doi.org/10.1128/mbio.01825-25","host_type":"journal"},{"url":"https://doi.org/10.1128/mbio.01825-25","host_type":"publisher"},{"url":"https://journals.asm.org/doi/pdf/10.1128/mbio.01825-25","host_type":"publisher"},{"url":"https://pubmed.ncbi.nlm.nih.gov/40985725","host_type":"repository"},{"url":"https://doaj.org/article/04c90ef04e414ae5a50cca1d1d919bea","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/12607567","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC12607567","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC12607567?pdf=render","host_type":"Europe_PMC"}],"fields_of_study":["Pneumocystis jirovecii pneumonia detection and treatment","Antifungal resistance and susceptibility","Cytomegalovirus and herpesvirus research","Extracellular Vesicles","Gene Editing","CRISPR-Cas Systems","Pneumocystis","Animals","Mice","Transformation, Genetic","Gene Transfer Techniques","Lung","Plasmids"],"mesh_terms":["Extracellular Vesicles","Gene Editing","Animals","Lung","Plasmids","Pneumocystis","Transformation, Genetic","Gene Transfer Techniques","Mice","CRISPR-Cas Systems"],"keywords":["Obligate","Genome editing","Transformation (genetics)","Gene","Plasmid","DNA","Model organism","Gene targeting","CRISPR","Electroporation","Functional genomics","Genetic manipulation","Mycology","Extracellular Vesicles","Pneumocystis","Antifungal Resistance","Homology-directed Repair","Crispr/cas9"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-06T01:40:14.749316Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}