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Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in Pneumocystis murina.
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 DNA cleavage efficiency in vitro. 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.IMPORTANCEPneumocystis 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 Pneumocystis murina, achieved through in vivo delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the dhps 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 Pneumocystis and other obligate, host-adapted microbes.
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Strategies of genome editing in mycobacteria: Achievements and challenges.
Tremendous amount of physiological and functional complexities acquired through decades of evolutionary pressure makes Mycobacterium tuberculosis (Mtb) one of the most dreadful microorganisms infecting humans from centuries. Astonishing advances in genomics and genome editing tools substantially grew our knowledge about Mtb as an organism but dramatically failed to completely understand it as a pathogen. Though conventional tools based on homologous recombination, antisense, controlled proteolysis, etc. have made important contributions in advancing our understanding of the pathophysiology of Mtb, yet these approaches have not accentuated our exploration of mycobacterium on account of certain technical limitations. In this review article we have compiled various approaches implemented in genome editing of mycobacteria along with the latest adaptation of clustered regularly interspaced short palindromic repeat (CRISPR)-interference (CRISPRi), emphasizing the achievements and challenges associated with these techniques.
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High-Efficiency Genome Editing in Naturally Isolated Aeromonas hydrophila and Edwardsiella Piscicida Using the CRISPR-Cas9 System.
Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR-Cas9 system for high-efficiency, marker-less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the aerA gene (encoding for aerolysin, a pore-forming toxin that plays a critical role in the pathogenicity) and the gfp insertion mutants in three naturally isolated A. hydrophila strains. Additionally, we deleted five putative hemolysin-encoding genes in both A. hydrophila ML10-51K and its ∆aerA derivative. The same system was also applied to the naturally isolated E. piscicida S11-285 strain, successfully deleting the ssaV gene (a component of the Type III Secretion System-a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies.
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CRISPR/Cas9 technology in the modeling of and evaluation of possible treatments for Niemann-Pick C.
Niemann-Pick disease type C (NPC) is a rare neurodegenerative condition resulted from mutations in NPC1 and NPC2 genes. This cellular lipid transferring disorder mainly involves endocytosed cholesterol trafficking. The accumulation of cholesterol and glycolipids in late endosomes and lysosomes results in progressive neurodegeneration and death. Recently, genome editing technologies, particularly CRISPR/Cas9 have offered the opportunity to create disease models to screen novel therapeutic options for this disorder. Moreover, these methods have been used for the purpose of gene therapy. This review summarizes the studies that focused on the application of CRISPR/Cas9 technology for exploring the mechanism of intracellular cholesterol transferring, and screening of novel agents for treatment of NPC.
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PASTE: The Way Forward for Large DNA Insertions.
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Prime editing in plants: prospects and challenges.
Prime editors are reverse transcriptase (RT)-based genome-editing tools that utilize double-strand break (DSB)-free mechanisms to decrease off-target editing in genomes and enhance the efficiency of targeted insertions. The multiple prime editors that have been developed within a short span of time are a testament to the potential of this technique for targeted insertions. This is mainly because of the possibility of generation of all types of mutations including deletions, insertions, transitions, and transversions. Prime editing reverses several bottlenecks of gene editing technologies that limit the biotechnological applicability to produce designer crops. This review evaluates the status and evolution of the prime editing technique in terms of the types of editors available up to prime editor 5 and twin prime editors, and considers the developments in plants in a systematic manner. The various factors affecting prime editing efficiency in plants are discussed in detail, including the effects of temperature, the prime editing guide (peg)RNA, and RT template amongst others. We discuss the current obstructions, key challenges, and available resolutions associated with the technique, and consider future directions and further improvements that are feasible to elevate the efficiency in plants.
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CRISPR/Cas as a tool to overcome drug resistance in cancer: From challenge to opportunity.
Drug resistance remains a significant challenge in cancer therapy, often resulting in treatment failure, tumor progression, and metastasis. The underlying resistance mechanisms-including genetic mutations, epigenetic alterations, and modifications in drug efflux pathways-are complex and not yet fully understood. This review explores the application of CRISPR-Cas gene editing technology in understanding and overcoming drug resistance in cancer. It focuses on how CRISPR can identify and target resistance-associated genes to restore drug sensitivity. CRISPR-based approaches enable precise genetic modifications that offer new insights into the molecular basis of drug resistance. The technology has shown promise in dissecting resistance mechanisms and developing targeted therapeutic strategies. Nevertheless, key limitations such as inefficient delivery systems, off-target effects, and limited specificity hinder clinical translation. Current efforts focus on improving guide RNA design, creating more effective delivery vectors, and integrating CRISPR with existing treatments. CRISPR-Cas technology holds significant potential to address drug resistance in cancer by enabling targeted genetic interventions. Continued advancements are required to enhance its safety, specificity, and delivery, paving the way for its integration into future clinical applications.
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Implementation of RAGATH RNA-associated DNA Endonucleases as Genome Editing Tool in Escherichia coli.
The preferred method for Escherichia coli genome editing relies on Cas9 from Streptococcus pyogenes (SpCas9) and λ-Red recombinase. Although SpCas9 is currently the most active RNA-guided DNA endonuclease, a significant number of escapers are often observed, making it inefficient across different sites, particularly when inserting large fragments. In this study, we identified two RAGATH RNA-associated DNA endonucleases (RADs) derived from IS607 transposons. Both of them exhibited high cleavage activity in E. coli. When combined with λ-Red recombinase, they achieved editing efficiencies approaching 100%. Even at target sites where SpCas9 exhibited low editing efficiency, RADs maintained efficiencies ranging from 57% to 94%. Moreover, RADs exhibited higher efficiencies in inserting large fragments in certain cases compared to SpCas9. Taken together, these RAD-based genome editing tools provide viable alternatives to SpCas9, particularly for challenging targets and/or large fragment insertions.
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Fine-Tuning Homology-Directed Repair (HDR) for Precision Genome Editing: Current Strategies and Future Directions.
CRISPR-Cas9 is a powerful genome-editing technology that can precisely target and cleave DNA to induce double-strand breaks (DSBs) at almost any genomic locus. While this versatility holds tremendous therapeutic potential, the predominant cellular pathway for DSB repair-non-homologous end-joining (NHEJ)-often introduces small insertions or deletions that disrupt the target site. In contrast, homology-directed repair (HDR) utilizes exogenous donor templates to enable precise gene modifications, including targeted insertions, deletions, and substitutions. However, HDR remains relatively inefficient compared to NHEJ, especially in postmitotic cells where cell cycle constraints further limit HDR. To address this challenge, numerous methodologies have been explored, ranging from inhibiting key NHEJ factors and optimizing donor templates to synchronizing cells in HDR-permissive phases and engineering HDR-enhancing fusion proteins. These strategies collectively aim to boost HDR efficiency and expand the clinical and research utility of CRISPR-Cas9. In this review, we discuss recent advances in manipulating the balance between NHEJ and HDR, examine the trade-offs and practical considerations of these approaches, and highlight promising directions for achieving high-fidelity genome editing in diverse cell types.
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Delivery of Targeting Constructs into Zygotes Using Electroporation.
Zygote electroporation (EP) is a widely used technique for the delivery of targeting constructs into the embryos and has become very popular in the last few years in transgenic facilities all over the world. Zygote electroporation is technically simple, as it does not require equipment for microinjection. Moreover, zygote electroporation is less invasive, resulting in higher survival rates in comparison to pronuclear microinjection, leading to a reduction in the number of animals needed. Another advantage of zygote electroporation is a higher number of zygotes that can be targeted simultaneously, which minimizes the time required for zygote manipulation. Zygote electroporation is easily combined with the use of the CRISPR-Cas9 system.
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