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Cell Reports Jun 2024Cyclic GMP-AMP synthase (cGAS) undergoes liquid-liquid phase separation (LLPS) to trigger downstream signaling upon double-stranded DNA (dsDNA) stimulation, and the...
Cyclic GMP-AMP synthase (cGAS) undergoes liquid-liquid phase separation (LLPS) to trigger downstream signaling upon double-stranded DNA (dsDNA) stimulation, and the condensed cGAS colocalizes with stress granules (SGs). However, the molecular mechanism underlying the modulation of cGAS activation by SGs remains elusive. In this study, we show that USP8 is localized to SGs upon dsDNA stimulation and potentiates cGAS-stimulator of interferon genes (STING) signaling. A USP8 inhibitor ameliorates pathological inflammation in Trex1 mice. Systemic lupus erythematosus (SLE) databases indicate a positive correlation between USP8 expression and SLE. Mechanistic study shows that the SG protein DDX3X promotes cGAS phase separation and activation in a manner dependent on its intrinsic LLPS. USP8 cleaves K27-linked ubiquitin chains from the intrinsically disordered region (IDR) of DDX3X to enhance its condensation. In conclusion, we demonstrate that USP8 catalyzes the deubiquitination of DDX3X to facilitate cGAS condensation and activation and that inhibiting USP8 is a promising strategy for alleviating cGAS-mediated autoimmune diseases.
Topics: Humans; Animals; Nucleotidyltransferases; Ubiquitin Thiolesterase; Mice; Ubiquitination; DEAD-box RNA Helicases; Interferon Type I; Stress Granules; Lupus Erythematosus, Systemic; Signal Transduction; Mice, Inbred C57BL; HEK293 Cells; Membrane Proteins; Mice, Knockout; Exodeoxyribonucleases; Endopeptidases; Phosphoproteins; Endosomal Sorting Complexes Required for Transport
PubMed: 38795350
DOI: 10.1016/j.celrep.2024.114248 -
International Journal of Molecular... May 2024The myostatin () gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent...
The myostatin () gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between and age-related diseases. It is unclear how and age-associated muscle loss in other animals are related. In this study, we utilized gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to and muscle cell senescence. The expression of was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that influenced three prime repair exonuclease 1 () expression through the SMAD2/3 complex. The downregulation of contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of in cell senescence in large animals.
Topics: Animals; Myostatin; Cattle; Cellular Senescence; Exodeoxyribonucleases; Signal Transduction; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphoproteins; Cells, Cultured
PubMed: 38791317
DOI: 10.3390/ijms25105277 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They... (Review)
Review
Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.
Topics: Gene Editing; CRISPR-Cas Systems; Transcription Activator-Like Effector Nucleases; Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Engineering; Humans
PubMed: 38783797
DOI: 10.13345/j.cjb.230615 -
Analytica Chimica Acta Nov 2023Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and...
BACKGROUND
Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and sensitive detection methods is vital to prevent and manage foodborne diseases. In this study, we used tetraethylenepentamine (TEPA)-functionalized magnetic nanoparticles (MNPs) and a loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a-based biosensor to concentrate and detect, respectively, L. monocytogenes. LAMP enables DNA amplification at a constant temperature, providing a highly suitable approach for point-of-care testing (POCT). The ability of CRISPR/Cas12a to cleave ssDNA reporter, coupled with TEPA-functionalized MNPs effective attachment to negatively charged bacteria, forms a promising biosensor.
RESULTS
The LAMP assay was meticulously developed by selecting specific primers and designing crRNA sequences targeting a specific region within the hly gene of L. monocytogenes. We selected primer and refined the amplification conditions by systematically exploring a temperature range from 59 °C to 69 °C, ensuring the attainment of optimal performance. This process was complemented by systematic optimization of LAMP-CRISPR/Cas12a system parameters. In particular, we successfully established the optimal ssDNA reporter concentrations (0-1.2 μM) and Cas12a-mediated trans-cleavage times (0-20 min), crucial components that underpin the effectiveness of the LAMP-CRISPR/Cas12a-based biosensor. For optimizing parameters in capturing L. monocytogenes using TEPA-functionalized MNPs, capture efficiency was significantly enhanced through adjustments in TEPA-functionalized MNPs concentration, incubation times, and magnetic separation duration. Large-volume (20 mL) magnetic separation exhibited a 10-fold sensitivity improvement over conventional methods. Utilizing TEPA-functionalized MNPs, the LAMP-CRISPR/Cas12a-based biosensor achieved detection limits of 10 CFU mL in pure cultures and 10 CFU g in enoki mushrooms.
SIGNIFICANCE
The integration of this novel technique with the LAMP-CRISPR/Cas12a-based biosensor enhances the accuracy and sensitivity of L. monocytogenes detection in foods, and it can be a promising biosensor for POCT. The 10-fold increase in sensitivity compared to conventional methods makes this approach a groundbreaking advancement in pathogenic bacteria detection for food safety and public health.
Topics: Listeria monocytogenes; Biosensing Techniques; Nucleic Acid Amplification Techniques; CRISPR-Cas Systems; Magnetite Nanoparticles; Limit of Detection; Food Microbiology; Bacterial Proteins; Endodeoxyribonucleases; Molecular Diagnostic Techniques; CRISPR-Associated Proteins
PubMed: 38783743
DOI: 10.1016/j.aca.2023.341905 -
Gut May 2024Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to...
OBJECTIVE
Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH.
DESIGN
C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated.
RESULTS
We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract).
CONCLUSION
Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.
PubMed: 38777573
DOI: 10.1136/gutjnl-2023-331447 -
Analytica Chimica Acta Jun 2024CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step...
BACKGROUND
CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA.
RESULTS
Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %.
SIGNIFICANCE
The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.
Topics: SARS-CoV-2; DNA, Single-Stranded; Nucleic Acid Amplification Techniques; Humans; RNA, Viral; COVID-19; Limit of Detection; CRISPR-Cas Systems; Endodeoxyribonucleases; CRISPR-Associated Proteins; Bacterial Proteins
PubMed: 38772660
DOI: 10.1016/j.aca.2024.342693 -
BMC Biology May 2024Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be...
BACKGROUND
Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging.
RESULTS
Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control.
CONCLUSIONS
Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.
Topics: Gene Editing; Humans; Exodeoxyribonucleases; CRISPR-Cas Systems; HEK293 Cells; DNA Repair Enzymes
PubMed: 38769511
DOI: 10.1186/s12915-024-01918-w -
Journal of Nanobiotechnology May 2024The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited...
The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited accessibility and specificity. This study proposes a rapid and convenient Cas12a-based assay for the direct detection of CEA in clinical serum samples, aiming to address these limitations. The protocol involves a rolling machine operation, followed by a 5-min Cas12a-mediated cleavage process. The assay demonstrates the capability to detect human serum with high anti-interference performance and a detection limit as low as 0.2 ng/mL. The entire testing procedure can be accomplished in 75 min without centrifugation steps, and successfully reduced the limit of detection of traditional DNA walking machine by 50 folds. Overall, the testing procedure can be easily implemented in clinical settings.
Topics: Carcinoembryonic Antigen; Humans; Biosensing Techniques; Limit of Detection; DNA; CRISPR-Cas Systems; Endodeoxyribonucleases; Nucleic Acid Amplification Techniques; CRISPR-Associated Proteins; Bacterial Proteins
PubMed: 38762451
DOI: 10.1186/s12951-024-02535-z -
The Journal of Biological Chemistry Jun 2024Bacterial RecJ exhibits 5'→3' exonuclease activity that is specific to ssDNA; however, archaeal RecJs show 5' or 3' exonuclease activity. The hyperthermophilic archaea...
Bacterial RecJ exhibits 5'→3' exonuclease activity that is specific to ssDNA; however, archaeal RecJs show 5' or 3' exonuclease activity. The hyperthermophilic archaea Methanocaldococcus jannaschii encodes the 5'-exonuclease MjRecJ1 and the 3'-exonuclease MjRecJ2. In addition to nuclease activity, archaeal RecJ interacts with GINS, a structural subcomplex of the replicative DNA helicase complex. However, MjRecJ1 and MjRecJ2 do not interact with MjGINS. Here, we report the structural basis for the inability of the MjRecJ2 homologous dimer to interact with MjGINS and its efficient 3' hydrolysis polarity for short dinucleotides. Based on the crystal structure of MjRecJ2, we propose that the interaction surface of the MjRecJ2 dimer overlaps the potential interaction surface for MjGINS and blocks the formation of the MjRecJ2-GINS complex. Exposing the interaction surface of the MjRecJ2 dimer restores its interaction with MjGINS. The cocrystal structures of MjRecJ2 with substrate dideoxynucleotides or product dCMP/CMP show that MjRecJ2 has a short substrate binding patch, which is perpendicular to the longer patch of bacterial RecJ. Our results provide new insights into the function and diversification of archaeal RecJ/Cdc45 proteins.
Topics: Archaeal Proteins; Crystallography, X-Ray; Methanocaldococcus; Protein Binding; Protein Multimerization; DNA Helicases; Models, Molecular; Exodeoxyribonucleases
PubMed: 38762184
DOI: 10.1016/j.jbc.2024.107379 -
Cell Reports May 2024Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every...
Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.
Topics: Animals; Macrophages; Cell Differentiation; DNA Damage; Phagocytosis; Drosophila Proteins; Signal Transduction; Caspases; Enzyme Activation; Deoxyribonucleases; Drosophila melanogaster; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases
PubMed: 38761374
DOI: 10.1016/j.celrep.2024.114251