Search Results (1,511)

Pneumonia caused by Pneumocystis jirovecii poses a serious threat, particularly to immunocompromised patients such as those with HIV/AIDS, transplant recipients, or individuals undergoing chemotherapy. Its diagnosis is challenging because current methods, such as microscopy and certain molecular tests, have limitations in sensitivity and specificity, and require specialized equipment, which delays treatment initiation. In this context, CRISPR-Cas12-based methods offer a promising alternative: they are rapid, highly specific, sensitive, and low-cost, enabling more timely and accessible detection, even in resource-limited settings. We developed a simple and rapid detection platform based on the CRISPR-Cas12 coupled with lateral flow strips. A guide RNA was designed against DHPS, β-tubulin, and mtLSU rRNA genes. The guide corresponding to β-tubulin showed high sensitivity in the detection of P. jirovecii to produce a detectable fluorescence signal within the first 20–30 min. In addition, it demonstrated high specificity for P. jirovecii when DNA from other microorganisms was used. When coupled with lateral flow strips, high sensitivity and specificity were also observed for detecting positive samples, without the need for genetic amplification. CRISPR-Cas12 successfully detected P. jirovecii infection in an initial diagnostic application, demonstrating the potential of this method for integration into public health diagnostic systems, particularly in field, due to its adaptability, speed, and ease of use. Full article

CRISPR/Cas systems have made significant progress in the field of molecular diagnostics in recent years. To overcome the aerosol contamination problem brought on by amplicon transfer in the common two-step procedure, the “one-pot method” has become a major research hotspot in this field. However, these methods usually rely on specially designed devices or additional chemical modifications. In this study, a novel “one-pot” strategy was developed to detect the foodborne pathogen Cronobacter sakazakii (C. sakazakii). A specific sequence was screened out from the virulence gene ompA of C. sakazakii as the detection target. Combining with the recombinase polymerase amplification (RPA), a rapid detection platform for C. sakazakii based on the CRISPR/Cas12a system was established for the first time. The sensitivity of this method was determined from three different levels, which are 10⁻⁴ ng/μL for genomic DNA (gDNA), 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture. Without any microbial enrichment, the detection limits for artificially contaminated cow and goat milk powder samples were 4.65 CFU/mL and 4.35 CFU/mL, respectively. To address the problem brought on by aerosol contamination in the common RPA-CRISPR/Cas12a two-step method, a novel pipette tip-in-tube (PTIT) method for simple and sensitive one-pot nucleic acid detection was further developed under the inspiration of the capillary principle. The RPA and CRISPR/Cas systems were isolated from each other by the force balance of the solution in a pipette tip before amplification. The detection limits of the PTIT method in pure bacterial culture and the spiked samples were exactly the same as that of the two-step method, but with no false positive cases caused by aerosol contamination at all. Compared with other existing one-pot methods, the PTIT method requires no additional or specially designed devices, or any chemical modifications on crRNA and nucleic acid probes. Therefore, the PTIT method developed in this study provides a novel strategy for realizing one-pot CRISPR/Cas detection easily and holds significant potential for the rapid point-on-care testing (POCT) application. Full article

Human papillomavirus (HPV) is a prime target for genome-editing therapy as its E6 and E7 oncogenes are crucial for cancer development and maintenance. A key challenge in CRISPR/Cas9 therapy is the off-target effects. This study utilized a double-nicking technique to introduce DNA breaks in the E6 and E7 regions of HPV16. From 146 gRNA candidates, 16 double-nicking pairs were selected. Multiple combinations of double-nicking (DN)-gRNA pairs were delivered to HPV16-positive cells via lentiviruses, followed by Cas9 nickase (Cas9n) expression. Combinations of 3–4 DN-gRNA pairs effectively killed HPV16-positive cells while sparing HPV-negative cells. Off-target effects were reduced by nearly three orders of magnitude. An “all-in-one” adenovirus (AdV) system expressing four gRNA pairs and Cas9n showed promise in inhibiting tumor growth in HPV16-positive cancer models, demonstrating its potential as a safe and effective treatment for HPV-induced tumors. Full article

Low-density lipoprotein receptor-related protein 5 (LRP5) is a multifunctional transmembrane coreceptor that plays a pivotal role in development and disease. Wnt/β-catenin signaling is the primary downstream signaling pathway activated by LRP5. Furthermore, some LRP5 functions are mediated by noncanonical pathways, such as AKT/P21 and TGF-β/Smad signaling. Pathologically, both loss-of-function and gain-of-function mutations in LRP5 produce distinct phenotypes, ranging from osteoporosis-pseudoglioma syndrome to high bone mass disorders. Beyond the skeletal system, LRP5 has emerged as a key regulator of retinal angiogenesis, vascular integrity, renal tubular function, neurodevelopment, and lipid metabolism. Its physiological functions are highlighted by its ability to influence adipocyte differentiation, insulin sensitivity, and neuronal synaptic plasticity. Moreover, LRP5 displays a dual role in development and disease progression. Although it plays a protective role in acute injuries such as myocardial infarction and acute kidney injury, LRP5 also contributes to chronic pathologies such as tubulointerstitial fibrosis, polycystic kidney disease, and atherosclerosis through fibrotic and inflammatory pathways. Recent therapeutic interest has focused on modulating LRP5 activity using agents such as anti-Dickkopf-related protein 1 antibody, sclerostin inhibitors, polyclonal antibodies, CRISPR/Cas9 knockout, and some natural products. This review discusses the current understanding of LRP5's physiological and pathological roles across organ systems and highlights its therapeutic potential, emphasizing the need for targeted approaches considering its context-dependent effects. Full article

Antimicrobial resistance (AMR) is a growing global health emergency that threatens the effectiveness of modern medicine, exacerbating healthcare costs, morbidity, and mortality, particularly in low- and middle-income countries (LMICs). Traditional approaches to antimicrobial development and stewardship have proven inadequate in curbing the rapid emergence and spread of resistant pathogens. This review explores cutting-edge biotechnological innovations as sustainable, precision-based solutions to combat AMR and promote global health equity. A comprehensive narrative review was conducted using literature published between 2018 and 2023 from PubMed, ScienceDirect, and Web of Science. Peer-reviewed studies focusing on novel antimicrobial strategies were thematically analyzed, with attention to efficacy, feasibility, and translational readiness. Key innovations identified include nanotechnology-enhanced antimicrobial delivery, bacteriophage therapy, CRISPR-Cas gene editing, immunotherapy, and personalized medicine. These strategies demonstrated substantial in vitro and in vivo efficacy, such as >90% MRSA biofilm reduction via silver nanoparticles and 95% carbapenem susceptibility restoration in E. coli using CRISPR-Cas9. When integrated with machine learning and rapid diagnostics, these approaches enable precision-targeted therapies and data-informed stewardship, offering scalable solutions adaptable to diverse healthcare systems. Antimicrobial resistance demands urgent, equitable innovation. Integrating biotechnologies like CRISPR, phage therapy, and nanomedicine with data-driven tools offers promising solutions. To ensure real-world impact, we recommend establishing regionally tailored translational research platforms and public–private partnerships as the most effective strategy to scale innovations and strengthen AMR response in low-resource settings. Full article

In the context of global climate change and efforts toward “carbon peak and carbon neutrality,” forest resource protection and restoration have become fundamental to ecological civilization. The genetic improvement of trees, as the primary component of forest ecosystems, holds strategic importance for ecological security, resource supply, and carbon neutrality. Traditional tree breeding techniques, including selective and hybrid breeding, have established robust technical systems through extensive practice. However, these methods face limitations such as extended cycles, reduced efficiency, and constrained genetic gains in meeting contemporary requirements. Modern biotechnologies, including genomic selection (GS), gene editing (CRISPR/Cas9), and marker-assisted selection (MAS), substantially enhance the precision and efficiency of genetic improvement. Nevertheless, exclusive reliance on either traditional or modern methods proves insufficient for addressing complex environmental adaptation and rapid breeding requirements. Consequently, the integration of traditional breeding with modern biotechnology to develop intelligent, sustainable, and efficient breeding strategies has emerged as a central focus in tree genetics and breeding. An integrated “step-by-step” approach warrants promotion, supported by a multi-source data sharing platform, an optimized core germplasm repository, and a “climate-soil-genotype” matching model to facilitate the region-specific deployment of improved varieties. Full article

Liposomes are lipid bilayer vesicles that are highly biocompatible, able to interact with the cell membrane, and able to release their cargo easily. The improvement of the physicochemical properties of liposomes, such as surface charge, lipid composition, and functionalization, makes these vesicles eligible delivery nanosystems for the gene therapy of many pathological conditions. In the present study, pre-formulation analysis was conducted to develop liposomes that facilitate the delivery of nucleic acids to neuronal cells, with the aim of future delivery of a CRISPR/Cas9 system designed to silence genes responsible for autosomal dominant neurodegenerative disorders. To this aim, different nucleic acid cargo models, including λ phage DNA, plasmid DNA, and mRNA encoding GFP, were considered. Liposomes with varying lipid compositions were produced using the ethanol injection method and analyzed for their dimensional stability and ability to interact with DNA. The selected formulations were tested in vitro using a neuroblastoma cell line (SH-SY5Y) to evaluate their potential toxicity and the ability to transfect cells with a DNA encoding the green fluorescent protein (pCMV-GFP). Among all formulations, the one containing phosphatidylcholine, phosphatidylethanolamine, pegylated 1,2-distearoyl-sn-glycero-3-phosphethanolamine, cholesterol, and dioctadecyl-dimethyl ammonium chloride (in the molar ratio 1:2:4:2:2) demonstrated the highest efficiency in mRNA delivery. Although this study was designed with the goal of ultimately enabling the delivery of a CRISPR/Cas9 system for treating autosomal dominant neurodegenerative disorders such as polyglutamine spinocerebellar ataxias (SCAs), CRISPR/Cas9 components were not delivered in the present work, and their application remains the objective of future investigations. Full article

Extracellular vesicles (EVs) are mediators of intercellular communication and serve as promising tools for drug discovery and targeted therapies. These lipid bilayer-bound nanovesicles facilitate the transfer of functional proteins, RNAs, lipids, and other biomolecules between cells, thereby influencing various physiological and pathological processes. This review outlines the molecular mechanisms governing EV biogenesis and cargo sorting, emphasizing the role of key regulatory proteins in modulating selective protein packaging. We explore the critical involvement of EVs in various disease microenvironments, including cancer progression, neurodegeneration, and immunological modulation. Their ability to cross biological barriers and deliver bioactive cargo makes them desirable candidates for precise drug delivery systems, especially in neurological and oncological disorders. Moreover, this review highlights advances in engineering EVs for the delivery of RNA therapeutics, CRISPR-Cas systems, and targeted small molecules. The utility of EVs as diagnostic tools in liquid biopsies and their integration into personalized medicine and companion diagnostics are also discussed. Patient-derived EVs offer dynamic insights into disease states and enable real-time treatment stratification. Despite their potential, challenges such as scalable isolation, cargo heterogeneity, and regulatory ambiguity remain significant hurdles. Recent studies have reported novel pharmacological approaches targeting EV biogenesis, secretion, and uptake pathways, with emerging regulators showing promise as drug targets for modulating EV cargo. Future directions include the standardization of EV analytics, scalable biomanufacturing, and the classification of EV-based therapeutics under evolving regulatory frameworks. This review emphasizes the multifaceted roles of EVs and their transformative potential as therapeutic platforms and biomarker reservoirs in next-generation precision medicine. Full article

Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression. Full article

Enteroviruses, a diverse genus within the Picornaviridae family, are responsible for a wide range of human infections, including hand, foot, and mouth disease, respiratory disease, aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis. Despite their substantial global health burden and the frequent emergence of outbreaks, no specific antiviral therapies are currently approved for clinical use against non-polio enteroviruses. This review provides a comprehensive overview of the current landscape of antiviral strategies targeting enteroviruses, including direct-acting antivirals such as capsid binders, protease inhibitors, and viral RNA polymerase inhibitors. We also examine the potential of host-targeting agents that interfere with virus–host interactions essential for replication. Emerging strategies such as immunotherapeutic approaches, RNA interference, CRISPR-based antivirals, and peptide-based antivirals are also explored. Furthermore, we address key challenges, including viral diversity, drug resistance, and limitations in preclinical models. By highlighting recent advances and ongoing efforts in antiviral development, this review aims to guide future research and accelerate the discovery of effective therapies against enterovirus infections. Full article

OCT4 is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of OCT4 by the transcription factor GATA4 is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of OCT4 with GATA4 in pigs. A CRISPR activation (CRISPRa) sgRNA library was designed and constructed, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. Then, a pig PK15 cell line was engineered with a single-copy OCT4 promoter-driven EGFP reporter at the ROSA26 locus, combined with the dCas9-SAM system for transcriptional activation. The CRISPRa sgRNA lentiviral library was used to screen for transcription factors, with or without GATA4 overexpression. Flow cytometry combined with high-throughput sequencing identified MYC, SOX2, and PRDM14 as activators and OTX2 and CDX2 as repressors of OCT4. In the presence of GATA4, transcription factors such as SALL4 and STAT3 showed synergistic activation. Functional validation confirmed that HOXD13 upregulates OCT4, while OTX2 inhibits it. GATA4 and SALL4 synergistically enhance OCT4 expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of OCT4 in pigs. Full article

Biofilms constitute a significant challenge in the therapy of infectious diseases, offering remarkable resistance to both pharmacological treatments and immunological elimination. This resilience is orchestrated through the regulation of extracellular polymeric molecules, metabolic dormancy, and quorum sensing, enabling biofilms to persist in both clinical and industrial environments. The resulting resistance exacerbates chronic infections and contributes to mounting economic burdens. This review examines the molecular and structural complexities that drive biofilm persistence and critically outlines the limitations of conventional diagnostic and therapeutic approaches. We emphasize advanced technologies such as super-resolution microscopy, microfluidics, and AI-driven modeling that are reshaping our understanding of biofilm dynamics and heterogeneity. Further, we highlight recent progress in biofilm-targeted therapies, including CRISPR-Cas-modified bacteriophages, quorum-sensing antagonists, enzyme-functionalized nanocarriers, and intelligent drug-delivery systems responsive to biofilm-specific cues. We also explore the utility of in vivo and ex vivo models that replicate clinical biofilm complexity and promote translational applicability. Finally, we discuss emerging interventions grounded in synthetic biology, such as engineered probiotic gene circuits and self-regulating microbial consortia, which offer innovative alternatives to conventional antimicrobials. Collectively, these interdisciplinary strategies mark a paradigm shift from reactive antibiotic therapy to precision-guided biofilm management. By integrating cutting-edge technologies with systems biology principles, this review proposes a comprehensive framework for disrupting biofilm architecture and redefining infection treatment in the post-antibiotic era. Full article

Increasingly complex epidemics of animal infectious diseases have emerged as a major risk to livestock production and human health. However, current detection methods for animal infectious diseases suffer from shortcomings such as insufficient sensitivity, complicated operation, and reliance on skilled personnel, highlighting the urgent need for novel sensing platforms. CRISPR/Cas systems are adaptive immune systems found in many prokaryotes. Owing to their ability to precisely and reliably target and cleave nucleic acids, the CRISPR/Cas-based nucleic acid detection technology is considered a promising new detection method. When leveraged with a pre-amplification step and established readout methods, CRISPR/Cas-based sensing platforms can achieve a high sensitivity of single-base resolution or attomolar levels on-site. In this review, we first outline the history, working principles, and nucleic acid detection platforms derived from various CRISPR/Cas systems. Next, we evaluate the advantages and limitations of different nucleic acid pre-amplification methods integrated with CRISPR/Cas systems, followed by a discussion of readout methods employed in CRISPR/Cas-based sensing platforms. Additionally, we highlight recent applications of CRISPR/Cas-based sensing platforms in identifying animal infectious diseases. Finally, we address the challenges and prospects of CRISPR/Cas-based sensing platforms for the early and accurate diagnosis of animal infectious diseases. Full article

Bioactive peptides encrypted in bovine β-casein display diverse physiological functions, including antihypertensive, antioxidative, antimicrobial, and immunomodulatory activities. These peptides are normally released during gastrointestinal digestion or microbial fermentation, especially by proteolytic systems of lactic acid bacteria (LAB). However, peptide yields vary widely among LAB strains, reflecting strain-specific protease repertoires. To overcome these limitations, the scientific goal of this study is to provide a comprehensive synthesis of how synthetic biology, molecular biotechnology, and systems-level approaches can be leveraged to enhance the targeted discovery and production of β-casein-derived bioactive peptides. Genome engineering tools such as clustered regularly interspaced short palindromic repeats associated system (CRISPR/Cas) systems have been applied to modulate gene expression and metabolic flux in LAB, while inducible expression platforms allow on-demand peptide production. Additionally, cell-free systems based on LAB lysates further provide rapid prototyping for high-throughput screening. Finally, multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, further help pinpoint regulatory bottlenecks and facilitate rational strain optimization. This review provides a comprehensive overview of bioactive peptides derived from bovine β-casein and highlights recent progress in LAB-based strategies—both natural and engineered—for their efficient release. These advances pave the way for developing next-generation functional fermented foods enriched with targeted bioactivities. Full article

Agrobacterium-mediated transformation systems are extensively applied in japonica rice varieties. However, the adaptability of local rice varieties to existing transformation systems remains limited, owing to their complex genotypes, posing a substantial challenge to transformation. In this study, four local rice varieties were selected to optimize the effects of different culture media on callus induction, browning resistance, contamination resistance, callus tolerance, differentiation, regeneration, and root development, and then two varieties were selected to improve plant architecture and tiller development by CRISPR/Cas9-mediated gene editing, based on constructive transformation systems. The goal was to enhance the transformation efficiency of local varieties and innovate germplasms. The results demonstrated that japonica rice varieties XG293 and WD68 exhibited higher induction rates under the treatment of 2 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) + 1 mg/L NAA (Naphthaleneacetic acid), whereas indica rice varieties H128 and E33 performed the best under 3 mg/L 2,4-D + 1 mg/L NAA. Severe browning in H128 was effectively mitigated by a carbon source of 20 g/L maltose supplemented with 40 mg/L ascorbic acid. Contamination after Agrobacterium infection was controlled by 300 mg/L Tmt (Timentin). Under a treatment of 200 µM/L acetosyringone +10 min infection duration, XG293 and WD68 exhibited higher callus tolerance, differentiation rates, and GUS staining rates, achieving transformation efficiencies of 43.24% and 52.38%, respectively. In contrast, H128 and E33 performed better under the treatment of 200 µM/L Acetosyringone + 5 min, with transformation efficiencies of 40.00% and 40.74%, respectively. The mutants after OsCCD7 gene editing in WD68 and H128 displayed a dwarfness of plant height, a significant increase in tiller numbers, and compact architecture. These findings demonstrate that an optimized combination of plant growth regulators and infection durations effectively improves transformation efficiency for local varieties, and the OsCCD7 gene regulates plant architecture and tiller development with variable effects, depending on the rice complex genotypes. This study provides a theoretical basis for the efficient transformation of local rice varieties and germplasm innovation. Full article