Search Results (3,518)

Air pollution has become one of the biggest problems throughout the world. Smog has a severe effect on the pulmonary and circulatory systems, which causes a significant number of deaths globally. Therefore, the remediation of air pollutants to maintain ecosystem processes and functions and to improve human health is a crucial problem confronting mankind today. This review aims to discuss the health effects of smog on humans. This review will also focus on the bioremediation of air pollution (smog) using bacteria, fungi, phytoremediation, nanotechnology, and phylloremediation (using plants and microbes). Phylloremediation is the most effective technology for removing air pollution naturally. The future perspective presents a great need to produce an ecosystem where microbes, plants, and nanoparticles synergistically control smog. In addition, further advancements would be needed to modify the genetic makeup of microbes and plants. Biotechnological approaches like CRISPR-Cas9 can be applied to the editing and cutting of specific genes responsible for the bioremediation of VOCs, NOx, SOx, and harmful hydrocarbons. The extracted genes can then be expressed in biologically modified microorganisms and plants for the enhanced bioremediation of smog. Full article

Rice (Oryza sativa L.), a globally staple food crop, frequently encounters growth, developmental, and yield limitations due to phosphate deficiency. LOW PHOSPHATE ROOT1/2 (LPR1/2) are essential genes in plants that regulate primary root growth and respond to local phosphate deficiency signals under low phosphate stress. In rice, five LPR genes, designated OsLPR1–OsLPR5 based on their sequence identity with AtLPR1, have been identified. OsLPR3 and OsLPR5 are specifically expressed in roots and induced by phosphate deficiency, contributing to rice growth, development, and the maintenance of phosphorus homeostasis under low phosphate stress. In contrast, OsLPR2 is uniquely expressed in shoots, suggesting it may have distinct functions compared with other family members. This study employed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) gene editing technology to generate oslpr2 mutant transgenic lines and subsequently investigated the effect of OsLPR2 gene knockout on rice growth, phosphate utilization, and salt stress tolerance in the seedling stage, as well as the effect of OsLPR2 gene knockout on rice development and agronomic traits in the maturation stage. The results indicated that the knockout of OsLPR2 did not significantly impact rice seedling growth or phosphate utilization, which contrasts significantly with its homologous genes, OsLPR3 and OsLPR5. However, the mutation influenced various agronomic traits at maturity, including plant height, tiller number, and seed setting rate. Moreover, the OsLPR2 mutation conferred enhanced salt stress tolerance in rice. These findings underscore the distinct roles of OsLPR2 compared with other homologous genes, establishing a foundation for further investigation into the function of the OsLPR family and the functional differentiation among its members. Full article

Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The Aspergillus flavus hacA gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the hacA gene at the DNA and transcript levels affected the survival, growth, and development of A. flavus. Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops. Full article

The duck embryo fibroblast cell line CCL-141, which is currently the only commercialized duck cell line, has been underexplored in adipogenesis research. (1) Background: This study establishes an experimental protocol to induce adipogenesis in CCL-141 cells, addressing the importance of understanding gene functions in this process. (2) Methods: Chicken serum, fatty acids, insulin, and all-trans retinoic acid were used to treat CCL-141 cells, with adipogenesis confirmed by Oil Red O staining and gene expression quantification. CRISPR/Cas9 technology was applied to knockout PPARγ, and the resulting adipogenic phenotype was assessed. (3) Results: The treatments promoted adipogenesis, and the knockout of PPARγ validated the cell line’s utility for gene function studies. (4) Conclusions: CCL-141 cells are a suitable model for investigating duck adipogenesis, contributing to the understanding of regulatory factors in this biological process. Full article

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management. Full article

Pine wilt disease (PWD), caused by the nematode Bursaphelenchus xylophilus, is a highly destructive forest disease that necessitates rapid and precise identification for effective management and control. This study evaluates various detection methods for PWD, including morphological diagnosis, molecular techniques, and remote sensing. While traditional methods are economical, they are limited by their inability to detect subtle or early changes and require considerable time and expertise. To overcome these challenges, this study emphasizes advanced molecular approaches such as real-time polymerase chain reaction (RT-PCR), droplet digital PCR (ddPCR), and loop-mediated isothermal amplification (LAMP) coupled with CRISPR/Cas12a, which offer fast and accurate pathogen detection. Additionally, DNA barcoding and microarrays facilitate species identification, and proteomics can provide insights into infection-specific protein signatures. The study also highlights remote sensing technologies, including satellite imagery and unmanned aerial vehicle (UAV)-based hyperspectral analysis, for their capability to monitor PWD by detecting asymptomatic diseases through changes in the spectral signatures of trees. Future research should focus on combining traditional and innovative techniques, refining visual inspection processes, developing rapid and portable diagnostic tools for field application, and exploring the potential of volatile organic compound analysis and machine learning algorithms for early disease detection. Integrating diverse methods and adopting innovative technologies are crucial to effectively control this lethal forest disease. Full article

This review article aims to summarize broadly recent developments in the treatment of HPV-associated cancers, including cervical cancer and head and neck squamous cell carcinoma. Relatively new treatments targeting the key HPV E6 and E7 oncoproteins, including gene editing with TALENs and CRISPR/Cas9, are discussed. Given the increased immunogenicity of HPV-related diseases, other therapies such as PRR agonists, adoptive cell transfer, and tumor vaccines are reaching the clinical trial phase. Due to the mechanism, immunogenicity, and reversibility of HPV carcinogenesis, HPV-related cancers present unique targets for current and future therapies. Full article

Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice. Full article

Deleterious variations in STXBP1 are responsible for early infantile epileptic encephalopathy type 4 (EIEE4, OMIM # 612164) because of its dysfunction in the central nervous system. The clinical spectrum of the neurodevelopmental delays associated with STXBP1 aberrations is collectively defined as STXBP1 encephalopathy (STXBP1-E), the conspicuous features of which are highlighted by early-onset epileptic seizures without structural brain anomalies. A girl was first diagnosed with unexplained disorders of movement and cognition, which later developed into STXBP1-E with unexpected leukoaraiosis and late onset of seizures. Genetic screening and molecular tests alongside neurological examinations were employed to investigate the genetic etiology and establish the diagnosis. A heterozygous mutation of c.37+2dupT at the STXBP1 splice site was identified as the pathogenic cause in the affected girl. The de novo mutation (DNM) did not result in any truncated proteins but immediately triggered mRNA degradation by nonsense-mediated mRNA decay (NMD), which led to the haploinsufficiency of STXBP1. The patient showed atypical phenotypes characterized by hypomyelinating leukodystrophy, and late onset of epileptic seizures, which had never previously been delineated in STXBP1-E. These findings strongly indicated that the haploinsufficiency of STXBP1 could also exhibit divergent clinical phenotypes because of the genetic heterogeneity in the subset of encephalopathies. Full article

CRISPR/Cas9 is a popular genome editing technology, yet its clinical application is hindered by off-target effects. Many deep learning-based methods are available for off-target prediction. However, few can predict off-target activities with insertions or deletions (indels) between single guide RNA and DNA sequence pairs. Additionally, the analysis of off-target data is challenged due to a data imbalance issue. Moreover, the prediction accuracy and interpretability remain to be improved. Here, we introduce a deep learning-based framework, named Crispr-SGRU, to predict off-target activities with mismatches and indels. This model is based on Inception and stacked BiGRU. It adopts a dice loss function to solve the inherent imbalance issue. Experimental results show our model outperforms existing methods for off-target prediction in terms of accuracy and robustness. Finally, we study the interpretability of this model through Deep SHAP and teacher–student-based knowledge distillation, and find it can provide meaningful explanations for sequence patterns regarding off-target activity. Full article

CRISPR/Cas9 has been applied to edit the genome of various organisms, but our understanding of editing outcomes at specific sites after Cas9-mediated DNA cleavage is still limited. Several deep learning-based methods have been proposed for repair outcome prediction; however, there is still room for improvement in terms of performance regarding frameshifts and model interpretability. Here, we present DeepIndel, an end-to-end multi-label regression model for predicting repair outcomes based on the BERT-base module. We demonstrate that our model outperforms existing methods in terms of accuracy and generalizability across various metrics. Furthermore, we utilized Deep SHAP to visualize the importance of nucleotides at various positions for DNA sequence and found that mononucleotides and trinucleotides in DNA sequences surrounding the cut site play a significant role in repair outcome prediction. Full article

Food safety is a major public health issue of global concern. In recent years, the CRISPR/Cas system has shown promise in the field of molecular detection. The system has been coupled with various nucleic acid amplification methods and combined with different signal output systems to develop a new generation of CRISPR/Cas-based nucleic acid biosensor technology. This review describes the design concept of the CRISPR/Cas-based nucleic acid biosensor and its application in food analysis. A detailed overview of different CRISPR/Cas systems, signal amplification methods, and signal output strategies is provided. CRISPR/Cas-based nucleic acid biosensors have the advantages of high sensitivity, strong specificity, and timeliness, achieving fast analysis of a variety of targets, including bacteria, toxins, metal ions, pesticides, veterinary drugs, and adulteration, promoting the development of rapid food safety detection technology. At the end, we also provide our outlook for the future development of CRISPR/Cas-based nucleic acid biosensors. Full article

The isolation of circulating tumoral DNA (ctDNA) present in the bloodstream brings about the opportunity to detect genomic aberrations from the tumor of origin. However, the low amounts of ctDNA present in liquid biopsy samples makes the development of highly sensitive techniques necessary to detect targetable mutations for the diagnosis, prognosis, and monitoring of cancer patients. Here, we employ standard genomic DNA (gDNA) and eight liquid biopsy samples from different cancer patients to examine the newly described CRISPR-Cas13a-based technology in the detection of the BRAF p.V600E actionable point mutation and appraise its diagnostic capacity with two PCR-based techniques: quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR). Regardless of its lower specificity compared to the qPCR and ddPCR techniques, the CRISPR-Cas13a-guided complex was able to detect inputs as low as 10 pM. Even though the PCR-based techniques have similar target limits of detection (LoDs), only the ddPCR achieved a 0.1% variant allele frequency (VAF) detection with elevated reproducibility, thus standing out as the most powerful and suitable tool for clinical diagnosis purposes. Our results also demonstrate how the CRISPR-Cas13a can detect low amounts of the target of interest, but its base-pair specificity failed in the detection of actionable point mutations at a low VAF; therefore, the ddPCR is still the most powerful and suitable technique for these purposes. Full article

Hysterothylacium aduncum is one of six pathogens responsible for human anisakiasis. Infection with H. aduncum can cause acute abdominal symptoms and allergic reactions and is prone to misdiagnosis in clinical practice. This study aims to enhance the efficiency and accuracy of detecting H. aduncum in food ingredients. We targeted the internal transcribed spacer 1 (ITS 1) regions of Anisakis to develop a visual screening method for detecting H. aduncum using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system. By comparing the ITS 1 region sequences of eight nematode species, we designed specific primers and CRISPR RNA (crRNA). The specificity of RPA primers was screened and evaluated, and the CRISPR system was optimized. We assessed its specificity and sensitivity and performed testing on commercial samples. The results indicated that the alternative primer ADU 1 was the most effective. The final optimized concentrations were 250 nM for Cas12a, 500 nM for crRNA, and 500 nM for ssDNA. The complete test procedure was achievable within 45 min at 37 °C, with a limit of detection (LOD) of 1.27 pg/μL. The amplified product could be directly observed using a fluorescence microscope or ultraviolet lamp. Detection results for 15 Anisakis samples were entirely consistent with those obtained via Sanger sequencing, demonstrating the higher efficacy of this method for detecting and identifying H. aduncum. This visual detection method, characterized by simple operation, visual results, high sensitivity, and specificity, meets the requirements for food safety testing and enhances monitoring efficiency. Full article

Descurainia sophia L. Webb ex Prantl is used in traditional medicine globally. However, the lack of an efficient and reliable genetic transformation system has seriously limited the investigation of gene function and further utilization of D. sophia. In this study, a highly efficient, time-saving, and cost-effective Agrobacterium tumefaciens-mediated genetic transformation system has been developed in D. sophia. In this method, the transformation was accomplished by simply dipping developing D. sophia inflorescences for 45 s into an Agrobacterium suspension (OD₆₀₀ = 0.6) containing 5% sucrose and 0.03% (v/v) Silwet L-77. Treated plants were allowed to set seeds which were then plated on a selective medium with hygromycin B (HygB) to screen transformants. Additionally, the CRISPR/Cas9 genomic editing system was validated by targeting phytoene desaturase (PDS) gene using this floral dip method, and mutant plants with the expected albino phenotype could be obtained in 2.5 months. This genetic transformation and targeted editing system will be a valuable tool for routine investigation of gene function and further exploitation in D. sophia. Full article