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At least 19 recordsLinked to original sources

PubMed2026

Cre-loaded integrase-defective lentiviral vectors for targeted cassette exchange in CHO cells.

Genome-modifying enzymes, such as recombinases and CRISPR-associated nucleases, enable targeted gene insertion when delivered transiently to minimize off-target effects. Precise genome engineering requires controlled enzyme activity, as well as efficient donor DNA transfer. Integrase-defective lentiviral vectors (IDLVs) provide a promising platform for transient episomal DNA transfer; however, their integration efficiency depends on complementary genome-targeting strategies. Here, we engineered Cre-loaded IDLVs (Cre-IDLVs) that co-package lentiviral vector genomes together with bioactive Cre recombinase. Cre was inserted into the Gag region of an integrase-defective gag-pol construct, allowing for efficient encapsidation and protease-mediated release during virion maturation without compromising the viral titer. The resulting particles carried donor cassettes flanked by heterospecific loxP sites. When applied to CHO founder cells harboring compatible genomic loxP landing pads, Cre-IDLVs efficiently mediated recombination-mediated cassette exchange, producing the highest number of G418-resistant colonies among the plasmid ratios tested. Genomic PCR and sequencing confirmed precise locus-specific insertion without detectable random integration in the analyzed clones. These findings establish Cre-IDLVs as a streamlined dual-delivery platform that couples transient recombinase activity with episomal donor DNA transfer. This hybrid lentiviral strategy provides a programmable approach for controlled and site-specific genome modification in mammalian cells.

Integrases↗

PubMed2026

Systematic profiling of nudivirus-like genes reveals conserved and differentiated roles in a domesticated endogenous virus.

Cotesia vestalis bracovirus (CvBV) is a type of domesticated endogenous virus (DEV) derived from ancestral nudiviruses that is integrated into the genome of the parasitoid wasp Cotesia vestalis. The CvBV proviral genome is composed of two distinct components: one encoding genes associated with virion morphogenesis and assembly, and the other harboring virulence genes that are excised, circularized, and packaged into virions. CvBV replication and particle assembly occur exclusively in the ovaries of female wasps. While prior studies have largely focused on the function of virulence genes during parasitization, the molecular mechanisms underlying CvBV replication and assembly remain poorly understood. Here, we identified 71 nudivirus-like genes in the C. vestalis genome through integrated transcriptomic and proteomic analyses. Using gene silencing and microscopy-based imaging approaches, we functionally characterized 24 key genes involved in DNA replication (helicase, integrase-1, and integrase-2), transcriptional regulation (p47, lef-5, and lef-9), capsid formation (vp39, PmV, HzNVorf9-1, HzNVorf9-2, HzNVorf106, 38k, 27b, and K425_459), envelope formation (11k, 17a-1, 35a-1, 35a-2, and K425_461), virion assembly (vlf-1, HzNVorf140-1, and HzNVorf140-2), and viral infectivity (pif-0 and vp91). Although the functions of most nudivirus-like genes are generally conserved among baculoviruses, nudiviruses, and bracoviruses, lef-5, K425_459, 11k, and vp91 appear to have undergone functional divergence relative to their homologs in baculoviruses, nudiviruses, and Microplitis demolitor bracovirus, highlighting lineage-specific adaptations in CvBV. Collectively, our work provides a molecular framework for understanding CvBV assembly and serves as a valuable resource for investigating bracovirus evolution.

Animals↗

PubMed2026

Building CRISPR immunity: evolution and mechanisms of spacer acquisition.

CRISPR-Cas systems in prokaryotes serve as adaptive immune systems that neutralize phage infections through RNA-guided nucleases. Immunization is achieved during the adaptation stage through Cas1-Cas2 integrase-mediated insertion of short foreign DNA snippets, termed spacers, into a CRISPR array in the host genome. This review examines the evolutionary origins of Cas1-Cas2 and the mechanisms of spacer acquisition in DNA-targeting CRISPR-Cas systems. Particular emphasis is placed on the recently characterized effector-assisted adaptation pathways, in which CRISPR effector proteins, such as Cascade and Cas9, typically involved in target interference, are repurposed for prespacer capture and integration into a CRISPR array.

CRISPR–Cas spacer acquisition↗

PubMed2026

Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Anhydrobiosis↗

PubMed2026

Genomic and transcriptomic characterization of genes expressed at 20 MPa by the marine actinobacterium Kocuria flava.

A marine hydrocarbonoclastic actinobacterium Kocuria flava IOS11 was isolated from 3500 m deep-sea water of the Indian Ocean. The isolate efficiently degraded phenanthrene (250 mg/L) achieving 82 and 98% of degradation at 0.1 MPa and 20 MPa, respectively within a period of 5 days. Whole genome, transcriptomee and metabolomic analysis elucidated its phenanthrene biodegradation efficiency under in situ deep-sea conditions. The genome sequence comprises 3.47 Mb distributed across 88 scaffolds with a high GC content of 74.30%. The genome analysis encoded 3126 genes including 3052 protein coding sequences with functional annotation identifying a broad array of genes associated with PAHs degradation, environmental stress adaptation, biosurfactant and siderophore synthesis. Transcriptome profiling under 0.1 and 20 MPa conditions with phenanthrene as a sole carbon source revealed enhanced expression of hydrocarbon degrading genes, transporters, biosurfactant associated enzymes and stress responsive genes including integrases, DNA repair protein Rad, alanine ligase, heat and cold shock proteins under high pressure conditions underscoring the deep-sea adaptation capabilities of the strain. The degradation pathway of phenanthrene was proposed through integrated genome, transcriptome and metabolomic analysis. These studies provided K. flava IOS11 as a metabolically versatile and pressure adapted bacterium with promising potential for bioremediation application in extreme marine environment.

Transcriptome↗

PubMed2024

Large language models improve annotation of prokaryotic viral proteins.

Viral genomes are poorly annotated in metagenomic samples, representing an obstacle to understanding viral diversity and function. Current annotation approaches rely on alignment-based sequence homology methods, which are limited by the paucity of characterized viral proteins and divergence among viral sequences. Here we show that protein language models can capture prokaryotic viral protein function, enabling new portions of viral sequence space to be assigned biologically meaningful labels. When applied to global ocean virome data, our classifier expanded the annotated fraction of viral protein families by 29%. Among previously unannotated sequences, we highlight the identification of an integrase defining a mobile element in marine picocyanobacteria and a capsid protein that anchors globally widespread viral elements. Furthermore, improved high-level functional annotation provides a means to characterize similarities in genomic organization among diverse viral sequences. Protein language models thus enhance remote homology detection of viral proteins, serving as a useful complement to existing approaches.

Viral Proteins↗

PubMed2026

Acquisition and erosion of toxin-antitoxin systems in bacterial chromosomes.

Toxin-antitoxin systems (TAs) are widespread in bacterial genomes. Yet, their integration, persistence, and impact in chromosome dynamics remain unclear. Here, we identified 80 type II TAs in the single chromosome of Photorhabdus laumondii TT01, 50 of which were experimentally validated. Comparative analysis across the Photorhabdus genus revealed a highly heterogeneous distribution, with TAs frequently clustering within discrete genomic regions, either alone or associated with cointegrate-forming transposases and integrases. TAs rarely clustered with other putative defense systems and are preferentially associated with different types of recombinases, suggesting distinct pathways of acquisition for the two types of functions. Functional analyses showed that most validated TAs display addictive properties and stabilize plasmids. These addictive TAs are preferentially located in genomic regions characterized by high gene turnover, consistent with recent acquisition events. Despite their plasmid-stabilizing capacity, TAs do not promote long-term conservation of their immediate chromosomal neighborhoods. Instead, we observed frequent TA loss, either through complete deletion or toxin pseudogenization, indicating relaxed selection for their persistence in bacterial lineages. We propose a stepwise model for TA evolution in bacterial chromosomes: initial acquisition mediated by mobile genetic elements, preferential integration into permissive genomic regions, subsequent genetic streamlining of linked loci, and progressive gene loss. The short-lasting linkage between TAs and their genomic neighborhoods is consistent with the view that TA modules can behave as autonomous, selfish genetic elements.

Journal Article↗

PubMed2023

Staphylococcus aureus Prophage-Encoded Protein Causes Abortive Infection and Provides Population Immunity against Kayviruses.

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

Humans↗

PubMed2026

Emergence and persistence of ESBL- and carbapenemase-producing Klebsiella pneumoniae-related species in Barcelona wastewater treatment plants.

The World Health Organization classifies extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing Klebsiella pneumoniae as critical-priority pathogens due to their high incidence, mortality, transmissibility, rapid resistance acquisition, and limited treatment options. Beyond clinical settings, their detection in wastewater treatment plants (WWTPs) provides an opportunity to assess their prevalence, persistence, and circulation within wastewater systems. This study characterized 37 antibiotic-resistant K. pneumoniae-related species strains isolated from two WWTPs in the metropolitan area of Barcelona, analyzing their antimicrobial resistance (AMR) profiles, antimicrobial resistance genes (ARGs), biocide and heavy metal tolerance genes (HMTGs), virulence factor genes (VFGs), biofilm-forming capacity, and conjugation ability. Among them, 70.3% were multidrug-resistant (MDR), and 16.2% were extensively drug-resistant. Whole-genome sequencing revealed diverse ARGs; all strains carried β-lactam resistance genes (14 ESBL and 12 carbapenemase producers), nearly all (96.9%) carried biocide or HMTGs, 64.9% harbored integrases, and all carried VFGs. Core-genome SNP analysis identified closely related strains across sampling periods and treatment stages, suggesting long-term persistence within the wastewater treatment system, despite biological and chemical processes in secondary treatment. Most strains (67.6%) displayed biofilm-forming capacity, and conjugation assays confirmed horizontal gene transfer in five of the seven ESBL-producing strains tested. High-risk clones were predominantly detected in the IFAS secondary treatment stage of the Gavà-Viladecans WWTP. The three strains recovered from the reclaimed water of the Baix Llobregat WWTP were ESBL or carbapenemase producers. Altogether, these results provide genomic and phenotypic evidence of the persistence and circulation of antibiotic-resistant K. pneumoniae-related species within wastewater treatment systems.IMPORTANCEWWTPs are essential for urban sanitation and environmental protection. Understanding how clinically relevant pathogens, such as ESBL and carbapenemase-producing K. pneumoniae-related species strains, behave in these settings may inform public health considerations. Investigating the presence and persistence of high-risk MDR pathogens in WWTPs helps identify circulation of AMR, assess the risk of gene transfer, and evaluate the potential for co-selection with other contaminants. This knowledge supports efforts to improve wastewater treatments, strengthen environmental surveillance, and develop integrated One Health strategies to limit the spread of AMR across human, animal, and environmental sectors.

Wastewater↗

PubMed2026

Chloramphenicol and tetracycline synergize with bacteriophage SeKF_13 to inactivate antimicrobial-resistant Salmonella Typhimurium.

UNLABELLED: Non-typhoidal Salmonella is estimated to cause up to 1 billion cases of global foodborne illness per year. Salmonella Typhimurium is a serovar of gravest worldwide concern as it is capable of infecting animal and human hosts and can also acquire antimicrobial resistance (AMR) determinants at a rapid rate. Recent advances in phage research have positioned them as especially useful for inactivation of Salmonella where antibiotics have proven no longer effective. Even more recently, phage-antibiotic synergy (PAS) has been proposed as a solution for AMR Salmonella, where synergistic combinations of phages and antibiotics are more effective than application of phage or antibiotic alone. Utilizing an in-house phage isolate, SeKF_13, we sought to determine the existence of PAS against a strain of Salmonella enterica serovar Typhimurium 14028 2a that is clinically resistant to bacteriostatic antibiotics chloramphenicol and tetracycline. Checkerboard assays revealed the presence of synergy when sub-lethal (sub-MIC) levels of either tetracycline or chloramphenicol were combined with phage SeKF_13 (P < 0.05; two-way ANOVA). Compared to tetracycline or chloramphenicol alone, the addition of phage also decreased the MICs of both antibiotics twofold. We also monitored the development of resistance and found that PAS significantly suppressed emergence of resistance compared to the antibacterial agents alone (P < 0.05; Tukey's HSD). Whole-genome sequencing revealed that SeKF_13 is devoid of genes encoding integrase, antimicrobial resistance, and virulence, ensuring safety in future applications. Together, our results suggest that combined treatment of phage and antibiotic can improve antimicrobial efficacy against antibiotic-resistant Salmonella enterica. IMPORTANCE: Salmonella enterica is a foodborne pathogen that causes one of the highest rates of foodborne illness worldwide. They are also capable of becoming resistant to antimicrobials very rapidly (i.e., antimicrobial resistance; AMR) due to their ability to acquire AMR determinants, undermining the effectiveness of current treatments. Bacteriophages (phages), viral predators of bacteria, have been proven to be effective in some cases, but recently, phage-antibiotic synergy has been proposed as a more effective solution than phages or antibiotics alone. We found this was, indeed, the case; using phage SeKF_13 and tetracycline or chloramphenicol (to which the Salmonella strain was resistant), we found that combination treatment was significantly more effective than either treatment alone. These results demonstrate that combined treatment of phage and antibiotic can bolster treatment efficacy against AMR Salmonella.

Salmonella typhimurium↗

PubMed2026

Isolation, identification, and genomic characterization of Staphylococcus aureus phage vB_SauL_202595 and its bacteriostatic application in dairy products.

Staphylococcus aureus is an important pathogen associated with bovine mastitis and dairy product contamination, posing economic and public health risks through the food chain. In this study, a temperate phage, vB_SauL_202595, was isolated from a dairy farm environmental sample using S. aureus SHZ-0127 as the host, and its biological characteristics, genomic features, and antibacterial activity in dairy matrices were evaluated. vB_SauL_202595 lysed 18 of 66 tested S. aureus strains, with a lysis susceptibility rate of 27.3%, including 5 highly susceptible strains, indicating a relatively limited host range. The optimal multiplicity of infection was 0.01, the latent period was approximately 30 min, and the burst size was approximately 316 PFU/cell. The phage remained stable at 4°C-37°C and pH 6-10. Genome analysis showed that vB_SauL_202595 belongs to the class Caudoviricetes, has a genome of 44,503 bp with 33.59% GC content, and encodes 63 predicted proteins. No typical antibiotic resistance genes or major virulence factors were detected; however, integrase and repressor genes were identified, supporting its temperate nature. vB_SauL_202595 inhibited S. aureus SHZ-0127 growth, reduced mature biofilm biomass, and decreased viable bacterial counts in milk and yogurt, with reductions of 1.23 and 1.42 log10 CFU/mL under representative conditions, respectively. From a One Health perspective, these findings provide foundational evidence for reducing S. aureus contamination and related antimicrobial resistance risks along the dairy chain. Overall, vB_SauL_202595 represents a candidate phage resource for dairy-associated S. aureus biocontrol research, but its limited host range and lysogeny-related genes require further safety assessment before food-related applications.IMPORTANCEStaphylococcus aureus is a major pathogen associated with bovine mastitis and a common contaminant in dairy products, causing economic losses and public health risks through the food chain. Although phage-based biocontrol has emerged as a promising strategy for controlling S. aureus contamination in dairy products, systematic evidence regarding phage activity in actual dairy matrices remains limited. In this study, we isolated and characterized a dairy farm environment-derived temperate phage, vB_SauL_202595, and evaluated its biological characteristics, genomic features, host range, stability, biofilm removal ability, and antibacterial performance in milk and yogurt. These findings provide foundational experimental evidence for phage-based dairy biocontrol against S. aureus. However, due to its limited host range and lysogeny-related genomic features, vB_SauL_202595 should be considered a candidate phage resource for further study. Broader validation, including phage-cocktail testing, long-term storage assays, product quality assessment, and regulatory safety evaluation, is needed before practical application.

Staphylococcus aureus↗

PubMed2025

Distribution and molecular characterization of integron classes from Escherichia coli and Klebsiella pneumoniae isolates in Sulaymaniyah province of Iraq.

UNLABELLED: The environmental pollution from the misuse of antimicrobial drugs is fueling selection pressure in bacteria, thereby exacerbating the threat to global health. In Iraq, the situation is made worse by the poor implementation of the World Health Organization's Global Antimicrobial Resistance and Use Surveillance System (WHO-GLASS). Consequently, this study aimed to increase surveillance of the spread of antimicrobial resistance in Sulaymaniyah, Iraq. A total of 296 Enterobacteriaceae comprising 147 Klebsiella pneumoniae and 149 Escherichia coli were isolated from humans, poultry, and dairy farms. The isolates were screened using multiplex PCR to assess the prevalence of the clinically important integron integrase (intI) classes and antimicrobial resistance genes (ARGs) of commonly used antibiotics. Remarkably, 81.14% of the isolates carried at least 2 ARGs, 10.47% intI1, and 3.72% intI2. No intI3 was detected. A total of 663 ARGs were identified using multiplex PCR in the two Enterobacteriaceae: beta-lactamase genes were 43%, tetracycline resistance genes 25.20%, sulfonamide resistance gene 16.10%, quinolone resistance gene 10.2%, and aminoglycoside resistance genes 5.7%. K. pneumoniae harbored more integrons and ARGs than E. coli, thus posing a higher antimicrobial resistance threat in this province. This study underscores the importance of implementing more stringent WHO-GLASS and antibiotic stewardship to end the multidrug resistance crisis in Iraq. IMPORTANCE: These data are about the prevalence of integrons and resistance genes, helping to fill a significant gap in global surveillance efforts. Results can be used by global health authorities and the World Health Organization to develop national and international antimicrobial resistance (AMR) control strategies. The study is important because integrons are key genetic platforms that capture and disseminate antibiotic resistance genes among bacteria. In addition, Escherichia coli and Klebsiella spp. are among the top causes of hospital- and community-acquired infections, especially urinary tract infections, bloodstream infections, and pneumonia. Therefore, it will be riskier when these bacteria have a high rate of integrons and resistance genes because it impedes treatments during infection. Another importance of this study is that the study was carried out in Iraq. Iraq, like many low- and middle-income countries, faces challenges with unregulated antibiotic use, leading to high rates of AMR.

Escherichia coli↗

PubMed2026

Antibiotic Resistance Genes in Dust from Kindergarten Environments: A Systematic Review of Occurrence, Diversity, Determinants, and Exposure Implications.

Kindergarten environments combine high microbial exposure with increased immunological vulnerability, yet antibiotic resistance genes (ARGs) in kindergarten dust remain poorly characterized. This systematic review synthesized evidence on the occurrence and potential health relevance of ARGs in kindergarten dust. Following PRISMA 2020 guidelines, PubMed, Scopus, and Web of Science were searched. Four studies from China, Hong Kong, and Norway (2018-2024) met the inclusion criteria. ARGs were detected in all kindergarten dust samples, indicating that dust is a consistent reservoir of antibiotic resistance determinants. A consensus resistome (classes detected in ≥2 studies) encompassed sulfonamide, macrolide-lincosamide-streptogramin B (MLSB), tetracycline, beta-lactam, aminoglycoside, and multidrug resistance genes; beta-lactam resistance genes were the only class reported in all four studies. Clinically important ARGs associated with last-resort antibiotics, including mecA, vanA, blaNDM, and mcr-5, were reported in three studies. Class 1 integron-integrase genes (intI1) frequently co-occurred with ARGs, suggesting potential horizontal gene transfer. Limited evidence indicated higher ARG abundance in urban and winter samples. One study reported antibiotic-resistant bacteria carrying resistance markers concordant with those in kindergarten dust in the urine of children attending the same facilities; however, this cross-sectional, single-site evidence is consistent with, but not sufficient to establish, a dust-to-child exposure pathway. The available evidence supports the plausibility that kindergarten dust may contribute to children's exposure to ARGs and ARG-carrying bacteria, but current studies do not establish causal transmission from dust to child colonization or infection. Standardized monitoring and longitudinal studies are needed to assess health risks and guide mitigation strategies in early childhood educational settings.

Dust↗

PubMed2025

How to Study Gene Expression and Gain of Function of Hoxb1 in Mouse Heart Development.

Anterior Hox genes are required for genetic identity and anterior posterior patterning of the second heart field (SHF), which contributes to the formation of the embryonic heart in vertebrates. Defective contribution of SHF cells to the arterial or venous pole of the heart is often associated with severe congenital heart defects. The mouse Cre-lox system allows the activation of expression of any gene of interest in restricted tissues. We developed a gain of function approach that relies on the use of a CAG transgene to ectopically activate Hoxb1 expression in SHF cardiac progenitor cells through specific Cre activation. Therefore, we generated a floxed transgenic mouse line, CAG-Hoxb1-eGFP, which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP expression. When induced within the anterior SHF lineage, we detected heart defects in mouse embryos such as right ventricular hypoplasia. Here, we describe the strategy for generating and genetically crossing this transgenic mouse line. We also provide detailed protocols for whole-mount embryo and paraffin section in situ RNAscope hybridization and X-gal staining allowing investigation of SHF contribution during heart development.

Animals↗

PubMed2026

A photoactivatable Cre-loxP system for spatiotemporal genetic manipulation in mouse taste buds.

Conventional genetic approaches, including global gene KO and conditional KO strategies such as the Cre-loxP system, have some limitations arising from systemic effects or insufficient temporal resolution. The recently developed photoactivatable Cre (PA-Cre) system may have a potential to improve spatiotemporal control of gene manipulation. In this study, we established and validated the feasibility of the PA-Cre system using taste buds as a model. We generated TRE-PA-Cre:R26-rtTA/tdTomato mice to evaluate blue-light-induced Cre recombinase activity. Through systematic optimization of illumination parameters, we found that a single session of blue-light-illumination resulted in limited recombination efficiency, whereas a multisession illumination strategy markedly increased recombination efficiency. To further assess the utility of the PA-Cre system for gene KO, we generated TRE-PA-Cre:R26-rtTA:Tas1r3-flox mice and targeted a taste-related gene Tas1r3. Genomic DNA quantitative PCR and reverse transcription-quantitative PCR both showed partial reductions in Tas1r3 at the DNA and mRNA levels, respectively. Behavioral assays further revealed a selective decrease in sensitivity to sweet and umami stimuli. Together, these findings demonstrate PA-Cre-mediated gene manipulation in taste buds and establish a practical optical activation paradigm, providing a high-spatiotemporal-resolution tool for investigating gene function in optically targeted regions.

Animals↗

PubMed2026

Generation and validation of a Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of disease-associated smooth muscle cell states.

BACKGROUND: Phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling and cardiovascular disease. Recent lineage-tracing and single-cell transcriptomic studies have identified secreted phosphoprotein 1 (SPP1) as a prominent marker associated with disease-associated VSMC states, particularly those linked to fibrotic remodeling and vascular calcification. However, the cellular origins and fate of SPP1-associated VSMC populations remain incompletely understood. METHODS AND RESULTS: We generated a novel Spp1-rSTOPr-Cre (Spp1Cre) knock-in mouse line in which Cre recombinase is expressed from the endogenous Spp1 locus following Dre-mediated excision of a rox-flanked transcriptional STOP cassette. Correct targeting of the knock-in allele was validated by internal, 5' junction, 3' junction, and long-range PCR analyses, as well as Sanger sequencing. To establish an intersectional lineage-tracing strategy, Spp1Cre mice were crossed with Myh11DreERT2 and Rosa26-RSR-LSL-tdTomato-LSL-eGFP reporter mice, enabling permanent labeling of VSMC-derived populations following activation of the endogenous Spp1 locus. Under physiological conditions, eGFP-positive cells were detected at low frequency within the vascular wall and were predominantly negative for the contractile markers ACTA2 and MYH11. As a proof-of-principle application, eGFP-positive cells markedly expanded within atherosclerotic lesions induced by AAV-PCSK9D377Y and high-fat diet feeding. These lineage-traced cells remained largely ACTA2- and MYH11-negative, consistent with a modulated phenotype. Notably, only a minority of eGFP-positive cells expressed SPP1 or fibronectin at the time of analysis, demonstrating the utility of permanent lineage tracing for tracking cells with a history of endogenous Spp1 activation during vascular remodeling. CONCLUSION: We report the generation and validation of a novel Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of VSMC-derived populations that have activated the endogenous Spp1 locus. This genetic resource provides a valuable platform for investigating the origin, fate, and phenotypic evolution of Spp1-associated VSMC populations during vascular remodeling and cardiovascular disease.

Animals↗

PubMed2026

Multichannel genomic recording of biological information with ENGRAM.

Molecular recording is an emerging paradigm for measuring biology over time. Enhancer-mediated genomic recording of activity in multiplex (ENGRAM) is a recently described synthetic biology circuit architecture that converts the transient activity of cis-regulatory elements (CREs) into stable genomic records that can be retrospectively recovered via DNA sequencing. Here we provide a step-by-step protocol for conducting ENGRAM experiments and analyzing the resulting data. We also describe key design considerations for ENGRAM recorders, summarize the strengths and limitations of ENGRAM, and highlight applications, including multiplex signal recording and high-throughput CRE screening. In contrast to other systems for DNA-based recording in mammalian systems, ENGRAM relies on prime editing-mediated insertions to record the activity of a given CRE, such that it is inherently multiplexable-for example, four-base-pair insertions can represent the activities of up to 256 distinct CREs. A further contrast lies with ENGRAM's compatibility with DNA Typewriter, which facilitates the capture of signal order. For users with basic skills in molecular biology, mammalian cell culture and DNA sequencing analysis, ENGRAM experiments can typically be completed within 5-6 weeks.

Genomics↗

PubMed2025

Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers.

Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results redout (Fluorescence vs FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.

Antibiotic Resistance Gene↗