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Extracellular Vesicle-Mediated Delivery of Genetic Material for Transformation and CRISPR/Cas9-based Gene Editing in
Steven G Sayson1,2, Alan Ashbaugh1,2, Lillian C Bauer3,4
1Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH.
Abstract:
Pneumocystis 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 Pneumocystis 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 Pneumocystis murina utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to Pneumocystis, we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful in vitro transformation and subsequent expression of mNeonGreen and DhpsARS in P. murina organisms. Subsequently, we established stable in vivo expression of mNeonGreen in mice infected with transformed P. murina for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the P. murina Dhps gene, confirming its in vitro cleavage efficiency. Ultimately, we achieved successful in vivo CRISPR/Cas9-mediated homologous recombination, precisely introducing a DhpsARS mutation into the P. murina genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in Pneumocystis, thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.
Insights
Researchers developed a new method using extracellular vesicles (EVs) to genetically modify Pneumocystis murina. This breakthrough enables stable gene expression and editing in Pneumocystis, advancing research into fungal pneumonia treatments.
Area of Science:
- Mycology and Infectious Diseases
- Molecular Biology and Genetics
- Biotechnology and Drug Discovery
Background:
- Pneumocystis species are fungal pathogens causing severe pneumonia, especially in immunocompromised individuals.
- Lack of effective genetic tools hinders understanding Pneumocystis biology and developing new therapies.
Purpose of the Study:
- To establish a novel method for stable transformation and CRISPR/Cas9-mediated genetic editing of Pneumocystis murina.
- To utilize engineered extracellular vesicles (EVs) as a delivery system for genetic material.
Main Methods:
- Engineered mouse lung extracellular vesicles (EVs) to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components.
- Demonstrated in vitro transformation and gene expression (mNeonGreen, DhpsARS) of P. murina.
- Validated in vivo stable gene expression and CRISPR/Cas9-mediated gene editing (Dhps gene targeting) in infected mice.
Main Results:
- Achieved successful in vitro transformation and expression of reporter genes in P. murina.
- Established stable in vivo expression of mNeonGreen in P. murina within infected mice for up to 5 weeks.
- Confirmed in vitro cleavage efficiency of the CRISPR/Cas9 system targeting the Dhps gene and successful in vivo homologous recombination introducing a DhpsARS mutation.
Conclusions:
- Established a foundational methodology for genetic manipulation in Pneumocystis using EVs and CRISPR/Cas9.
- This approach enables functional genomics, drug target validation, and the generation of genetically modified Pneumocystis strains.
- Opens new avenues for advanced research and potential therapeutic applications against Pneumocystis pneumonia.
