RESUMEN

Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.

Asunto(s)

Terapia Genética , Isquemia , Lípidos , Polímeros , Animales , Isquemia/terapia , Terapia Genética/métodos , Lípidos/química , Ratones , Polímeros/química , Nanopartículas/química , Factor de Crecimiento de Hepatocito/genética , Línea Celular , Transfección/métodos , Plásmidos/genética , Técnicas de Transferencia de Gen , Masculino , Miembro Posterior/irrigación sanguínea , Catalasa/metabolismo

RESUMEN

The problem of bacterial resistance caused by antibiotic abuse is seriously detrimental to global human health and ecosystem security. The two-dimensional nanomaterial (2D) such as black phosphorus (BP) is recently expected to become a new bacterial inhibitor and has been widely used in the antibacterial field due to its specific physicochemical properties. Nevertheless, the effects of 2D-BP on the propagation of antibiotic resistance genes (ARGs) in environments and the relevant mechanisms are not clear. Herein, we observed that the sub-inhibitory concentrations of 2D-BP dramatically increased the conjugative transfer of ARGs mediated by the RP4 plasmid up to 2.6-fold at the 125 mg/L exposure level compared with the untreated bacterial cells. Nevertheless, 2D-BP with the inhibitory concentration caused a dramatic decrease in the conjugative frequency. The phenotypic changes revealed that the increase of the conjugative transfer caused by 2D-BP exposure were attributed to the excessive reactive oxygen species and oxidative stress, and increased bacterial cell membrane permeability. The genotypic evidence demonstrated that 2D-BP affecting the horizontal gene transfer of ARGs was probably through the upregulation of mating pair formation genes (trbBp and traF) and DNA transfer and replication genes (trfAp and traJ), as well as the downregulation of global regulatory gene expression (korA, korB, and trbA). In summary, the changes in the functional and regulatory genes in the conjugative transfer contributed to the stimulation of conjugative transfer. This research aims to broaden our comprehension of how nanomaterials influence the dissemination of ARGs by elucidating their effects and mechanisms.

Asunto(s)

Antibacterianos , Farmacorresistencia Bacteriana , Transferencia de Gen Horizontal , Fósforo , Plásmidos , Plásmidos/genética , Fósforo/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Conjugación Genética , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Nanoestructuras , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Bacterias/genética , Bacterias/efectos de los fármacos

RESUMEN

While conducting genomic surveillance of carbapenemase-producing Enterobacteriaceae (CPE) from patient colonisation and clinical infections at Birmingham's Queen Elizabeth Hospital (QE), we identified an N-type plasmid lineage, pQEB1, carrying several antibiotic resistance genes, including the carbapenemase gene bla KPC-2. The pQEB1 lineage is concerning due to its conferral of multidrug resistance, its host range and apparent transmissibility, and its potential for acquiring further resistance genes. Representatives of pQEB1 were found in three sequence types (STs) of Citrobacter freundii, two STs of Enterobacter cloacae, and three species of Klebsiella. Hosts of pQEB1 were isolated from 11 different patients who stayed in various wards throughout the hospital complex over a 13 month period from January 2023 to February 2024. At present, the only representatives of the pQEB1 lineage in GenBank were carried by an Enterobacter hormaechei isolated from a blood sample at the QE in 2016 and a Klebsiella pneumoniae isolated from a urine sample at University Hospitals Coventry and Warwickshire (UHCW) in May 2023. The UHCW patient had been treated at the QE. Long-read whole-genome sequencing was performed on Oxford Nanopore R10.4.1 flow cells, facilitating comparison of complete plasmid sequences. We identified structural variants of pQEB1 and defined the molecular events responsible for them. These have included IS26-mediated inversions and acquisitions of multiple insertion sequences and transposons, including carriers of mercury or arsenic resistance genes. We found that a particular inversion variant of pQEB1 was strongly associated with the QE Liver speciality after appearing in November 2023, but was found in different specialities and wards in January/February 2024. That variant has so far been seen in five different bacterial hosts from six patients, consistent with recent and ongoing inter-host and inter-patient transmission of pQEB1 in this hospital setting.

Asunto(s)

Brotes de Enfermedades , Plásmidos , beta-Lactamasas , Humanos , Plásmidos/genética , beta-Lactamasas/genética , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/epidemiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Klebsiella pneumoniae/efectos de los fármacos , Proteínas Bacterianas/genética , Enterobacter cloacae/genética , Enterobacter cloacae/aislamiento & purificación , Enterobacter cloacae/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Infección Hospitalaria/microbiología , Antibacterianos/farmacología , Citrobacter freundii/genética , Citrobacter freundii/aislamiento & purificación , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Hospitales , Enterobacter

RESUMEN

Background: Hemorrhoids are common conditions at or around the anus, to which numerous people suffer worldwide. Previous research has suggested that microbes may play a role in the development of hemorrhoids, and the origins of these microbes have been preliminarily investigated. However, no detailed research on the microbes related to hemorrhoid patients has been conducted. This work aims to provide an initial investigation into the microbes related to hemorrhoid patients with high quality whole genome sequencing. Methods: Forty-nine bacterial strains were isolated from seven hemorrhoid patients. Third-generation nanopore sequencing was performed to obtain high quality whole genome sequences. The presence of plasmids, particularly new plasmids, along with antibiotic resistance genes, was investigated for these strains. Phylogenetic analysis and genome comparisons were performed. Results: Out of the 31 plasmids found in the strains, 15 new plasmids that have not been observed previously were discovered. Further structural analysis revealed new multidrug-resistant conjugative plasmids, virulent plasmids, and small, high-copy mobile plasmids that may play significant functional roles. These plasmids were found to harbor numerous integrases, transposases, and recombinases, suggesting their ability to quickly obtain genes to change functions. Analysis of antibiotic resistance genes revealed the presence of antibiotic resistant-integrons. Together with the surprising number of new plasmids identified, as well as the finding of transmission and modification events for plasmids in this work, we came to the suggestion that plasmids play a major role in genetic plasticity. Conclusion: This study reveals that the diversity of plasmids in human-associated microbes has been underestimated. With the decreasing cost of whole-genome sequencing, monitoring plasmids deserves increased attention in future surveillance efforts.

Asunto(s)

Bacterias , Hemorroides , Filogenia , Plásmidos , Humanos , Plásmidos/genética , Hemorroides/microbiología , Hemorroides/genética , Bacterias/genética , Bacterias/aislamiento & purificación , Secuenciación Completa del Genoma , Masculino , Farmacorresistencia Bacteriana Múltiple/genética , Femenino , Adulto

RESUMEN

Tigecycline is a last-resort drug used to treat serious infections caused by multidrug-resistant bacteria. tet(X4) is a recently discovered plasmid-mediated tigecycline resistance gene that confers high-level resistance to tigecycline and other tetracyclines. Since the first discovery of tet(X4) in 2019, it has spread rapidly worldwide, and as a consequence, tigecycline has become increasingly ineffective in the clinical treatment of multidrug-resistant infections. In this study, we identified and analyzed tet(X4)-positive Escherichia coli isolates from duck farms in Hunan Province, China. In total, 976 samples were collected from nine duck farms. Antimicrobial susceptibility testing and whole-genome sequencing (WGS) were performed to establish the phenotypes and genotypes of tet(X4)-positive isolates. In addition, the genomic characteristics and transferability of tet(X4) were determined based on bioinformatics analysis and conjugation. We accordingly detected an E. coli strain harboring tet(X4) and seven other resistance genes in duck feces. Multi-locus sequence typing analysis revealed that this isolate belonged to a new clone, and subsequent genetic analysis indicated that tet(X4) was carried in a 4608-bp circular intermediate, flanked by ISVsa3-ORF2-abh elements. Moreover, it exhibited transferability to E. coli C600 with a frequency of 10-5. The detection of tet(X4)-harboring E, coli strains on duck farms enhances our understanding of tigecycline resistance dynamics. The transferable nature of the circular intermediate of tet(X4) contributing to the spread of tigecycline resistance genes poses a substantial threat to healthcare. Consequently, vigilant monitoring and proactive measures are necessary to prevent their spread.

Asunto(s)

Antibacterianos , Patos , Infecciones por Escherichia coli , Escherichia coli , Granjas , Pruebas de Sensibilidad Microbiana , Plásmidos , Tigeciclina , Secuenciación Completa del Genoma , Patos/microbiología , Tigeciclina/farmacología , Animales , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Antibacterianos/farmacología , China , Plásmidos/genética , Infecciones por Escherichia coli/veterinaria , Infecciones por Escherichia coli/microbiología , Heces/microbiología , Tipificación de Secuencias Multilocus , Farmacorresistencia Bacteriana Múltiple/genética , Genotipo , Enfermedades de las Aves de Corral/microbiología , Proteínas de Escherichia coli/genética , Transferencia de Gen Horizontal , Farmacorresistencia Bacteriana/genética

RESUMEN

Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing tools have transformed the landscape of genome editing. However, the absence of a robust and safe CRISPR delivery method continues to limit its potential for therapeutic applications. Despite the emergence of various methodologies aimed at addressing this challenge, issues regarding efficiency and editing operations persist. We introduce a microfluidic gene delivery system, called droplet cell pincher (DCP), designed for highly efficient and safe genome editing. This approach combines droplet microfluidics with cell mechanoporation, enabling encapsulation and controlled passage of cells and CRISPR systems through a microscale constriction. Discontinuities created in cell and nuclear membranes upon passage facilitate the rapid CRISPR-system internalization into the nucleus. We demonstrate the successful delivery of various macromolecules, including mRNAs (~98%) and plasmid DNAs (~91%), using this platform, underscoring the versatility of the DCP and leveraging it to achieve successful genome engineering through CRISPR-Cas9 delivery. Our platform outperforms electroporation, the current state-of-the-art method, in three key areas: single knockouts (~6.5-fold), double knockouts (~3.8-fold), and knock-ins (~3.8-fold). These results highlight the potential of our platform as a next-generation tool for CRISPR engineering, with implications for clinical and biological cell-based research.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Humanos , Microfluídica/métodos , Técnicas de Transferencia de Gen , Electroporación/métodos , Células HEK293 , Plásmidos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética

RESUMEN

This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

Asunto(s)

Clonación Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Plásmidos/genética , Clonación Molecular/métodos , Mutagénesis Sitio-Dirigida/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN/genética

RESUMEN

Plasmids are extrachromosomal replicons which can quickly spread resistance and virulence genes between clinical pathogens. From the tens of thousands of currently available plasmid sequences we know that overall plasmid diversity is structured, with related plasmids sharing a largely conserved 'backbone' of genes while being able to carry very different genetic cargo. Moreover, plasmid genomes can be structurally plastic and undergo frequent rearrangements. So, how can we quantify plasmid similarity? Answering this question requires practical efforts to sample natural variation as well as theoretical considerations of what defines a group of related plasmids. Here we consider the challenges of analysing and rationalising the current plasmid data deluge to define appropriate similarity thresholds.

Asunto(s)

Plásmidos , Plásmidos/genética , Variación Genética , Filogenia , Bacterias/genética , Bacterias/clasificación

RESUMEN

BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Methanococcus , Methanococcus/genética , Edición Génica/métodos , Plásmidos/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Genoma Arqueal , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética/métodos

RESUMEN

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Citrobacter freundii , Infecciones por Enterobacteriaceae , Fosfomicina , beta-Lactamasas , Humanos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Citrobacter freundii/genética , Citrobacter freundii/enzimología , Citrobacter freundii/efectos de los fármacos , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Enterobacteriaceae/microbiología , Fosfomicina/farmacología , Italia/epidemiología , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Secuenciación Completa del Genoma

RESUMEN

Episomal AMA1-based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, the gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next-generation AMA1-based plasmids that ensure homogeneous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5-fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10-fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi.

Asunto(s)

Aspergillus nidulans , Sistemas CRISPR-Cas , Plásmidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Plásmidos/genética , Expresión Génica , Ingeniería Metabólica/métodos , Vías Biosintéticas/genética , Productos Biológicos/metabolismo

RESUMEN

As one of the most widely used pesticides in the global fungicide market, tebuconazole has become heavily embedded in soil along with antibiotic resistance genes (ARGs). However, it remains unclear whether the selective pressure produced by tebuconazole affects ARGs and their horizontal transfer. In this experiment, we simulated a tebuconazole-contaminated soil ecosystem and observed changes in the abundance of ARGs and mobile genetic element (MGEs) due to tebuconazole exposure. We also established a plasmid RP4-mediated conjugative transfer system to investigate in depth the impact of tebuconazole on the horizontal transfer of ARGs and its mechanism of action. The results showed that under tebuconazole treatment at concentrations ranging from 0 to 10 mg/L, there was a gradual increase in the frequency of plasmid conjugative transfer, peaking at 10 mg/L which was 7.93 times higher than that of the control group, significantly promoting horizontal transfer of ARGs. Further analysis revealed that the conjugative transfer system under tebuconazole stress exhibited strong ability to form biofilm, and the conjugative transfer frequency ratio of biofilm to planktonic bacteria varied with the growth cycle of biofilm. Additionally, scanning electron microscopy and flow cytometry demonstrated increased cell membrane permeability in both donor and recipient bacteria under tebuconazole stress, accompanied by upregulation of ompA gene expression controlling cell membrane permeability. Furthermore, enzyme activity assays indicated significant increases in CAT, SOD activity, and GSH content in recipient bacteria under tebuconazole stress. Moreover, expression levels of transmembrane transporter gene trfAp as well as genes involved in oxidative stress and SOS response were found to be correlated with the frequency of plasmid conjugative transfer.

Asunto(s)

Biopelículas , Fungicidas Industriales , Transferencia de Gen Horizontal , Triazoles , Triazoles/toxicidad , Triazoles/farmacología , Fungicidas Industriales/toxicidad , Fungicidas Industriales/farmacología , Biopelículas/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Plásmidos/genética , Genes Bacterianos

RESUMEN

Synthetic pyrethroids are widely used insecticides which may cause chronic diseases in non-target organisms upon long-term exposure. Microbial degradation offers a reliable method to remove them from the environment. This study focused on Brevibacillus parabrevis BCP-09 and its enzymes for degrading pyrethroids. The predicted deltamethrin-degrading genes phnA and mhpC were used to construct recombinant plasmids. These plasmids, introduced into Escherichia coli BL21(DE3) cells and induced with L-arabinose. The results indicated that the intracellular crude enzyme efficiently degraded deltamethrin by 98.8 %, ß-cypermethrin by 94.84 %, and cyfluthrin by 73.52 % within 24 h. The hydrolytic enzyme MhpC possesses a catalytic triad Ser/His/Asp and a typical "Gly-X-Ser-X-Gly" conservative sequence of the esterase family. Co-cultivation of induced E. coli PhnA and E. coli MhpC resulted in degradation rates of 41.44 ± 3.55 % and 60.30 ± 4.55 %, respectively, for deltamethrin after 7 d. This study states that the degrading enzymes from B. parabrevis BCP-09 are an effective method for the degradation of pyrethroids, providing available enzyme resources for food safety and environmental protection.

Asunto(s)

Brevibacillus , Nitrilos , Piretrinas , Piretrinas/metabolismo , Brevibacillus/metabolismo , Brevibacillus/genética , Nitrilos/metabolismo , Insecticidas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolasas/metabolismo , Hidrolasas/genética , Biodegradación Ambiental , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Plásmidos/genética

RESUMEN

BACKGROUND: Due to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses. RESULTS: In animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates. CONCLUSIONS: Overall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.

Asunto(s)

Escherichia coli Enterohemorrágica , Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Animales , Marcadores Genéticos , Infecciones por Escherichia coli/veterinaria , Infecciones por Escherichia coli/microbiología , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/aislamiento & purificación , Humanos , Plásmidos/genética , Simulación por Computador , Bovinos , Reacción en Cadena de la Polimerasa/veterinaria , Ovinos , Islas Genómicas/genética

RESUMEN

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3' terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Genoma , Cabras , Animales , Cabras/genética , Edición Génica/métodos , Animales Modificados Genéticamente , Plásmidos/genética

RESUMEN

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.

Asunto(s)

Virus de la Fiebre Aftosa , Virus de la Fiebre Aftosa/genética , Animales , Línea Celular , Genoma Viral/genética , Replicación Viral , Fiebre Aftosa/virología , Cricetinae , Plásmidos/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Eliminación de Gen , Endopeptidasas

RESUMEN

Motivation: Mobile genetic elements (MGEs) are as ubiquitous in nature as they are varied in type, ranging from viral insertions to transposons to incorporated plasmids. Horizontal transfer of MGEs across bacterial species may also pose a significant threat to global health due to their capability to harbor antibiotic resistance genes. However, despite cheap and rapid whole-genome sequencing, the varied nature of MGEs makes it difficult to fully characterize them, and existing methods for detecting MGEs often do not agree on what should count. In this manuscript, we first define and argue in favor of a divergence-based characterization of mobile-genetic elements. Results: Using that paradigm, we present skandiver, a tool designed to efficiently detect MGEs from whole-genome assemblies without the need for gene annotation or markers. skandiver determines mobile elements via genome fragmentation, average nucleotide identity (ANI), and divergence time. By building on the scalable skani software for ANI computation, skandiver can query hundreds of complete assemblies against >65 000 representative genomes in a few minutes and 19 GB memory, providing scalable and efficient method for elucidating mobile element profiles in incomplete, uncharacterized genomic sequences. For isolated and integrated large plasmids (>10 kb), skandiver's recall was 48% and 47%, MobileElementFinder was 59% and 17%, and geNomad was 86% and 32%, respectively. For isolated large plasmids, skandiver's recall (48%) is lower than state-of-the-art reference-based methods geNomad (86%) and MobileElementFinder (59%). However, skandiver achieves higher recall on integrated plasmids and, unlike other methods, without comparing against a curated database, making skandiver suitable for discovery of novel MGEs. AVAILABILITY AND IMPLEMENTATION: https://github.com/YoukaiFromAccounting/skandiver.

Asunto(s)

Secuencias Repetitivas Esparcidas , Programas Informáticos , Genoma Bacteriano , Elementos Transponibles de ADN/genética , Plásmidos/genética

RESUMEN

Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Phytophthora , Plásmidos , Polietilenglicoles , Transformación Genética , Phytophthora/genética , Phytophthora/patogenicidad , Plásmidos/genética , Polietilenglicoles/farmacología , Edición Génica/métodos , Bosques , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/parasitología

RESUMEN

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Asunto(s)

Clostridium butyricum , Plásmidos , Clostridium butyricum/genética , Plásmidos/genética , Humanos , Expresión Génica , Biotecnología/métodos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Celulasa/genética , Celulasa/metabolismo , Genes Reporteros , Microbiología Industrial/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos

RESUMEN

Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Genoma Bacteriano , Plásmidos , Synechococcus , Edición Génica/métodos , Synechococcus/genética , Plásmidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen