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PubMed2026

EMTscore infers divergent EMT pathways from omics data and enables rapid screening for EMT-associated gene sets.

MOTIVATION: Quantitative analyses of epithelial-mesenchymal transition (EMT) have been widely used in several areas of biomedical sciences due to its importance in development and cancer progression, but its multi-contextual nature requires standardization and implementation of gene set scoring methods beyond capacities of conventional tools. RESULTS: We developed EMTscore, a package that provides an efficient implementation of unbiased scoring methods for multiple EMT pathways using individual single-cell or bulk omics data, and the package allows rapid screening for cellular processes correlated with EMT. AVAILABILITY AND IMPLEMENTATION: EMTscore is available from GitHub https://github.com/wenmm/EMTscore under the GNU General Public License, and is uploaded on Zenodo with a DOI 10.5281/zenodo.19487376.

Epithelial-Mesenchymal Transition↗

PubMed2025

Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide.

Bacteria secrete extracellular membrane vesicles (EMVs). Physiological functions and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the details of the molecular basis of EMV biogenesis have not yet been fully elucidated. In our previous work, an N-terminus-substituted FAAV peptide labeled with nitrobenzoxadiazole (NBD; nFAAV5-NBD) was developed. This peptide can sense the curvature of a lipid bilayer and selectively bind to EMVs even in the presence of cells. Here, we applied nFAAV5-NBD to a genome-wide screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. We analyzed the transposon insertion sites in hyper- and hypo-vesiculation mutants and identified 16 and six genes, respectively, with a transposon inserted within or near them. Targeted gene-disrupted mutants of the identified genes showed that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase β-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, d-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study demonstrates the utility of a novel screening method using a curvature-sensing peptide for mutants with altered EMV productivity and provides information on the genes related to EMV production.IMPORTANCEConventional methods for isolation and quantification of extracellular membrane vesicles (EMVs) are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying various genes associated with EMV production of Shewanella vesiculosa HM13, which would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be valuable for EMV applications, such as secreting useful substances as EMV cargoes and producing artificially functionalized EMVs.

Shewanella↗

PubMed2026

Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus.

In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.

Animals↗

PubMed2026

A homogeneous immunoassay based on AlphaLICA technology for detecting florfenicol residues in animal-derived foods.

Florfenicol (FF), a broad-spectrum amide antibiotic widely used in livestock, poultry, and aquaculture, poses potential threats to food safety and public health due to its residual accumulation. In this study, a novel homogeneous immunoassay based on Amplified Luminescent Proximity Homogeneous Assay (AlphaLICA) technology was developed for the first time for rapid screening of FF residues in milk and egg matrices. By covalently immobilizing the FF-BSA conjugate and goat anti-mouse IgG onto luminescent and photosensitive microspheres, respectively, the method achieved wash-free, homogeneous quantitative detection through a competitive immunoreaction. Under optimized conditions, the assay exhibited a linear range of 0.2-16.2 ng mL-1, with a limit of detection of 9.7 pg mL-1 and a limit of quantification of 183 pg mL-1. The intra- and inter-batch coefficients of variation ranged from 3.08% to 5.70% and 2.44% to 7.09%, respectively. Spike recovery rates in milk and egg matrices ranged from 93.18% to 107.17% (RSD ≤ 5.57%). Cross-reactivity with 11 other common antibiotics, including chloramphenicol and thiamphenicol, was below 0.1%, demonstrating excellent specificity. Comparative analysis with a commercial ELISA kit showed high consistency (r2 = 0.9332, p < 0.001). With high sensitivity, strong specificity, simple operation, and a detection time of only 10 min, this method provides a reliable technical platform for high-throughput, rapid monitoring of FF residues in milk and egg matrices.

Journal Article↗

PubMed2025

Experimental strategy for characterization of novel TnpB orthologs.

TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.

Humans↗

PubMed2024

Targeting peptide antigens using a multiallelic MHC I-binding system.

Identifying highly specific T cell receptors (TCRs) or antibodies against epitopic peptides presented by class I major histocompatibility complex (MHC I) proteins remains a bottleneck in the development of targeted therapeutics. Here, we introduce targeted recognition of antigen-MHC complex reporter for MHC I (TRACeR-I), a generalizable platform for targeting peptides on polymorphic HLA-A*, HLA-B* and HLA-C* allotypes while overcoming the cross-reactivity challenges of TCRs. Our TRACeR-MHC I co-crystal structure reveals a unique antigen recognition mechanism, with TRACeR forming extensive contacts across the entire peptide length to confer single-residue specificity at the accessible positions. We demonstrate rapid screening of TRACeR-I against a panel of disease-relevant HLAs with peptides derived from human viruses (human immunodeficiency virus, Epstein-Barr virus and severe acute respiratory syndrome coronavirus 2), and oncoproteins (Kirsten rat sarcoma virus, paired-like homeobox 2b and New York esophageal squamous cell carcinoma 1). TRACeR-based bispecific T cell engagers and chimeric antigen receptor T cells exhibit on-target killing of tumor cells with high efficacy in the low nanomolar range. Our platform empowers the development of broadly applicable MHC I-targeting molecules for research, diagnostic and therapeutic applications.

Humans↗

PubMed2026

Benchmarking Assembly-Free K-mer Methods for Species Identification in Complex Plant Groups: A Case Study in Populus.

Species identification in taxonomically complex plant groups is frequently limited by the inadequacy of organellar markers, whose phylogenetic signal is disrupted by cytonuclear discordance and chloroplast capture. Using the taxonomically complex genus Populus as a model, we evaluated an assembly-free k-mer workflow against a curated SNP reference benchmark. Whole-genome resequencing data from 235 Populus individuals were curated to a 202-individual, 34-species reference dataset in which all retained species are strictly monophyletic in a genome-wide SNP analysis. Independent maximum likelihood analyses further confirmed that the 31 non-hybrid backbone species each maintained high-support monophyly, while taxa of documented reticulate origin showed placement patterns consistent with their reticulate histories. ABBA-BABA D-statistics detected widespread residual allele sharing within the backbone, though the strongest signals did not correspond to the species pairs responsible for the few k-mer identification failures. Against this benchmark, complete plastomes showed limited resolution, recovering only 3.0% species monophyly and 71.1% nearest-neighbor assignment. The optimized k-mer workflow, operating directly on raw reads without assembly or alignment, recovered 91.2% species monophyly, 99.0% nearest-neighbor assignment, and 98.0% group-average assignment. K-mer length was the primary accuracy-controlling parameter, with k = 31 falling within a stable accuracy plateau. Distance-based metrics reached near-saturation at 0.2× sequencing depth, indicating that low-coverage genome skimming can support scalable nuclear genome-based identification with standard computational resources. K-mer distance heatmaps also flagged unusual genomic affinities in hybrid-origin and outlier samples, providing a rapid screen for subsequent population genomic analyses. These results support assembly-free k-mer distances as an efficient tool for reference-based species identification and sample screening in complex plant groups, with residual limitations concentrated near recently diverged species boundaries. Model-based phylogenomic, coalescent, and network analyses remain necessary for resolving deeper species relationships and detailed introgression histories.

Populus↗

PubMed2026

Development and evaluation of a one-pot RPA-Cas12a assay based on a primer-driven reverse screening strategy for preliminary screening of megalocytivirus-related viruses.

A primer-driven reverse-screening strategy was used to identify an RPA-Cas12a target suitable for the rapid preliminary screening of megalocytivirus-related viruses. The ISKNV reference genome NC_003494.1 was used as the initial template, and candidate amplification units were designed according to RPA primer-design requirements, primer physicochemical properties, and the availability of Cas12a protospacer-adjacent motif (PAM) sites and crRNA target sequences. Following preliminary amplification assessment, the retained candidate primers were aligned individually against 75 complete genome sequences of megalocytivirus-related viruses. Of these, 67 sequences met the predefined criteria for target-region integrity, primer-binding-site compatibility, and Cas12a recognition. Retrospective mapping to the reference genome located the candidate amplification region within ORF057L. Based on the resulting candidate detection unit, a one-pot RPA-Cas12a assay incorporating a commercially available lyophilized RPA amplification module was developed. Optimization showed that 400 nM reporter and 80 nM crRNA-1 provided relatively stable fluorescence output. A cut-off value of 1281.6 relative fluorescence units (RFU) was established as the mean plus three standard deviations of the endpoint fluorescence values obtained from 20 qPCR-negative samples. In analytical sensitivity testing, the assay generated fluorescence signals above the negative control at low plasmid copy numbers. However, because only a limited number of replicates were tested at these low template concentrations, these findings were not used to define a formal limit of detection. ISKNV, RSIV, and TRBIV samples tested positive, whereas the MRV sample produced an endpoint fluorescence value below the cut-off. Repeatability analysis of the same sample in six independent reactions yielded a coefficient of variation of 8.03%. Among the 39 samples examined, no discordant qualitative results were observed between the RPA-Cas12a assay and qPCR. These findings support the use of the ORF057L-targeted one-pot RPA-Cas12a assay as a rapid preliminary screening tool for megalocytivirus-related viruses. Nevertheless, its formal limit of detection, inter-batch stability, cross-reactivity with additional non-target pathogens, and clinical diagnostic performance require further evaluation.

Lyophilized RPA↗

PubMed2026

Rapid glycomic analysis of serum EVs reveals altered N-glycosylation patterns in ASD.

Objective laboratory diagnostics for autism spectrum disorder (ASD) are lacking, necessitating rapid clinical screening tools. Because serum extracellular vesicle (EV) N-glycosylation captures critical neurodevelopmental signatures, we developed a fast, biologically interpretable diagnostic strategy. EVs from ASD patients with language impairment and neurotypical controls were isolated using a rapid extra-polyethylene glycol precipitation/filtration (EPF) workflow, benchmarked against ultracentrifugation. Following MALDI-TOF/MS profiling, machine learning was re-evaluated using repeated nested cross-validation to reduce optimistic bias and potential information leakage. Among five classifiers, Random Forest (RF) showed the best overall balance across discrimination, calibration, and classification metrics. RF-based SHAP analysis provided transparent interpretation, highlighting key discriminative glycans, including H4N3S1F1, H5N5S1F1, and H3N5F1. To elucidate molecular mechanisms, we integrated public EV transcriptomic data. This revealed significant dysregulation of N-glycosylation machinery genes (e.g., MAN1A1, NEU1, OSTC, RPN2), whose expression directionally aligned with observed glycan shifts in synaptic pathways. Collectively, this rapid serum EV N-glycomic workflow, combined with leakage-controlled RF-based interpretation, provides a promising foundation for non-invasive ASD biomarker discovery and future multicenter validation.

Humans↗

PubMed2026

Barcoded mutant library enables high-throughput functional genomics in a filamentous fungus.

Advances in sequencing technology enabling rapid and inexpensive whole-genome sequencing highlight how few genes are functionally characterized. This problem is particularly acute in filamentous fungi, where even in the best studied organisms upward of half of genes are poorly characterized or unannotated. High-throughput tools to identify gene function exist for single-celled organisms, like yeast and bacteria. However, filamentous fungi present challenges to high-throughput gene characterization, including low transformation efficiency and multinucleate cells. Filamentous fungi are critical components of nutrient cycling in ecosystems, form symbioses with plants that improve nutrient uptake, and are devastating human, plant, and animal pathogens causing millions of deaths and substantial crop loss each year. Thus, it is critical to overcome challenges to rapid gene characterization in filamentous fungi. We generated a library of hundreds of millions of uniquely barcoded plasmids containing a broad host-range drug resistance marker for ectopic insertion into filamentous fungal genomes by Agrobacterium tumefaciens. We then optimized A. tumefaciens mediated transformation of the biocontrol agent Trichoderma atroviride and made an insertional mutagenesis library containing 83,311 barcoded insertions, disrupting 5,331 of 11,863 predicted genes. This library enables high-throughput screens to rapidly connect genotype to phenotype. Quantifying relative barcode abundance in the pooled library before and after exposure to experimental conditions identified candidate genes and recovered known pathway components in amino acid biosynthetic, fructose utilization, and xylose utilization pathways. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling investigations of fungal biology to improve medical outcomes, biotechnology, and sustainable agriculture.

Genomics↗

PubMed2024

CRISPRi screens identify the lncRNA, LOUP, as a multifunctional locus regulating macrophage differentiation and inflammatory signaling.

Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP [lncRNA originating from upstream regulatory element of SPI1 (also known as PU.1)], as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate-determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames capable of being translated and are responsible for LOUP's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.

RNA, Long Noncoding↗

PubMed2024

Phenotypic targeting using magnetic nanoparticles for rapid characterization of cellular proliferation regulators.

Genome-wide CRISPR screens have provided a systematic way to identify essential genetic regulators of a phenotype of interest with single-cell resolution. However, most screens use live/dead readout of viability to identify factors of interest. Here, we describe an approach that converts cell proliferation into the degree of magnetization, enabling downstream microfluidic magnetic sorting to be performed. We performed a head-to-head comparison and verified that the magnetic workflow can identify the same hits from a traditional screen while reducing the screening period from 4 weeks to 1 week. Taking advantage of parallelization and performance, we screened multiple mesenchymal cancer cell lines for their dependency on cell proliferation. We found and validated pan- and cell-specific potential therapeutic targets. The method presented provides a nanoparticle-enabled approach means to increase the breadth of data collected in CRISPR screens, enabling the rapid discovery of drug targets for treatment.

Humans↗

PubMed2025

Active- and Allosteric-Site Cyclic Peptide Inhibitors of Secreted M. tuberculosis Chorismate Mutase.

The secreted Chorismate mutase enzyme of Mycobacterium tuberculosis (*MtbCM) is an underexplored potential target for the development of new antitubercular agents that are increasingly needed as antibiotic resistance rises in prevalence. As an enzyme suspected to be involved in virulence and host-pathogen interactions, disruption of its function could circumvent the difficulty of treating tuberculosis-infected granulomas. Drug development, however, is limited by novel ligand discovery. Currently, *MtbCM activity is measured by using a low throughput acid/base-mediated product derivatization absorbance assay. Here, we utilized an RNA-display affinity selection approach enabled by the Random Peptides Integrated Discovery (RaPID) system to screen a vast library of macrocyclic peptides (MCP) for novel *MtbCM ligands. Peptides identified from the RaPID selection, and analogs thereof identified by analyzing the selection population dynamics, produced a new class of *MtbCM inhibiting MCPs. Among these were two noteworthy "chorismides", whose binding modes were elucidated by X-ray crystallography. Both were potent inhibitors of the CM enzyme activity. One was identified as an allosteric binding peptide revealing a novel inhibition approach, while the other is an active-site binding peptide that when conjugated to a fluorescent probe allowed for the development of a series of alternative fluorescence-based ligand-displacement assays that can be utilized for the assessment of potential *MtbCM inhibitors.

Mycobacterium tuberculosis↗

PubMed2026

AI-driven CRISPR screening: optimizing gene editing through automation and intelligent decision support.

BACKGROUND: CRISPR-based genetic screening has become a central methodology in functional genomics, enabling systematic interrogation of gene function, genetic interactions and context-dependent vulnerabilities at scale. However, the rapid expansion of screening modalities-including multi-condition designs, combinatorial perturbations, in vivo applications and single-cell readouts-has exposed fundamental limitations of heuristic-driven experimental design and post hoc statistical analysis. MAIN BODY: This Review synthesizes how artificial intelligence is reshaping CRISPR screening by introducing predictive, adaptive and system-level intelligence across the experimental lifecycle. We organize recent advances into two tightly coupled modules. First, machine learning and deep learning (ML/DL) methods optimize experimental design by learning context-dependent perturbation behavior, anticipating confounding effects and enabling iterative, information-efficient screening strategies. Second, large language model-agent (LLM-agent) systems complement these advances by externalizing scientific reasoning, integrating biological knowledge at scale and coordinating analysis and decision-making in human-in-the-loop workflows. CONCLUSIONS: Together, ML/DL and LLM-agent approaches reframe CRISPR screening from a static analytical pipeline into an intelligent experimental system, with important implications for robustness, scalability and biological discovery.

Artificial Intelligence↗

PubMed2026

KCFtools: rapid alignment-free method for introgression screening and GWAS using k-mer profiles.

MOTIVATION: In the era of multiple genome references, researchers often align sequencing reads against distinct assemblies or even multiple references simultaneously. This enables applications such as the detection of introgressed segments or highly variable genomic regions, which are especially prevalent in large-genome crop species such as lettuce or wheat. However, these applications come at the cost of increased computational burden, inconsistencies in mapping methods, and reduced reproducibility across studies. To address these limitations, we developed KCFtools, a Java-based toolkit that identifies the presence and absence of k-mers in nonoverlapping genomic or transcriptomic windows by comparing query and reference genomes. This alignment-free approach enables the efficient computation of an identity score for each window, thereby facilitating robust detection of introgressed or variable regions across genomes. RESULTS: We systematically evaluated the performance and accuracy of the k-mer-based method implemented in KCFtools, benchmarking it against conventional single nucleotide variation-based introgression detection pipelines. Our results demonstrate that KCFtools effectively captures introgressed segments and structurally diverse regions, even in species with fragmented or highly divergent reference genomes. In addition, we extended KCFtools to generate genotype matrices from k-mer variation tables. These matrices are compatible with genome-wide association studies software and allow the identification of loci associated with phenotypic traits. We showcase the utility of this approach by detecting known and novel associations for downy mildew resistance in lettuce, underscoring the pipeline's potential for high-resolution, reference-agnostic population genetic analysis. AVAILABILITY AND IMPLEMENTATION: https://github.com/sivasubramanics/kcftools.

Software↗

PubMed2025

Methods for Cas13a expression and purification for use in CRISPR diagnostics.

The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.

SARS-CoV-2↗

PubMed2004

High-throughput method for detecting genomic-deletion polymorphisms.

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.

DNA Probes↗

PubMed2025

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗