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Bioelectrochemistry (Amsterdam,... Oct 2024Carcinoembryonic antigen (CEA), a key colon biomarker, demands a precise detection method for cancer diagnosis and prognosis. This study introduces a novel...
Triblock polyadenine-based electrochemical aptasensor for ultra-sensitive detection of carcinoembryonic antigen via exonuclease III-assisted target recycling and hybridization chain reaction.
Carcinoembryonic antigen (CEA), a key colon biomarker, demands a precise detection method for cancer diagnosis and prognosis. This study introduces a novel electrochemical aptasensor using a triblock polyadenine probe for ultra-sensitive detection of CEA. The method leverages Exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction. The triblock polyadenine probe self-assembles on the bare gold electrode through the strong affinity between adenine and gold electrode, blocking CEA diffusion and providing a large immobilization surface. CEA binding to hairpin probe 1 (HP1), followed by the hybridization between HP1 and hairpin probe 2 (HP2), triggers DNA cleavage by Exo III, amplifying the signal via a hybridization chain reaction and producing numerous dsDNA walkers that generates a dramatic electrochemical impedance signal. Under optimized conditions, the aptasensor achieved two ultra-low detection limits: 0.39 ag∙mL within the concentration range of 5 ag∙mL to 5 × 10 ag∙mL, and 1.5 ag∙mL within the concentration range of 5 × 10 ag∙mL to 1 × 10 ag∙mL. Its performance in human serum samples meets the practical standards, offering a promising new tool for ultrasensitive tumor marker detection, potentially revolutionizing early cancer diagnosis.
Topics: Exodeoxyribonucleases; Carcinoembryonic Antigen; Humans; Nucleic Acid Hybridization; Biosensing Techniques; Electrochemical Techniques; Aptamers, Nucleotide; Limit of Detection; Poly A; Gold; Electrodes
PubMed: 38823375
DOI: 10.1016/j.bioelechem.2024.108749 -
ACS Synthetic Biology Jun 2024Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To...
Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To alleviate these PAM constraints, protein engineering efforts have been applied to expand the PAM recognition range. In this study, we designed and constructed 990 synthetic hybrid Cas12a chimeras through domain shuffling and screened an efficient hybrid Cas12a (ehCas12a) that could recognize a broad range PAM of 5'-TYYN-3' (Y is T or C and N is A, T, C, or G). Furthermore, we constructed an ehCas12a variant, ehCas12a RRVR (T167R/N572R/K578V/N582R), with expanded PAM preference to 5'-TNYN, TWRV-3' (W is A or T, R is A or G, and V is A, C, or G), which can efficiently recognize -2* A/G PAMs that are barely recognized by Cas12a-type proteins and their mutants. Finally, we demonstrated that the DNase-inactivated ehCas12a RRVR base editor (dehCas12a RRVR-BE) was capable of targeting noncanonical PAMs and disease-related loci for potential therapeutic applications. Overall, our findings highlight the modular design and reconfiguration of Cas proteins for enhanced functionality.
Topics: Gene Editing; CRISPR-Cas Systems; CRISPR-Associated Proteins; Endodeoxyribonucleases; Bacterial Proteins; Protein Engineering; Humans; Escherichia coli
PubMed: 38819403
DOI: 10.1021/acssynbio.4c00103 -
PLoS Biology May 2024The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the...
The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Topics: Dependovirus; Humans; Gene Editing; Genetic Vectors; CRISPR-Cas Systems; Animals; HEK293 Cells; Genetic Therapy; CRISPR-Associated Proteins; Mice; Endodeoxyribonucleases; Bacterial Proteins
PubMed: 38814985
DOI: 10.1371/journal.pbio.3002619 -
Nature Communications May 2024The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization...
The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.
Topics: Animals; Transcription Factors; DNA Helicases; Nuclear Proteins; Mice; Nucleosomes; Indoleacetic Acids; RNA Polymerase II; Fibroblasts; Gene Knock-In Techniques; Hepatocytes; E1A-Associated p300 Protein; Transcriptional Activation; Transcription, Genetic; Histones; Deoxyribonuclease I; Chromatin; Humans
PubMed: 38811575
DOI: 10.1038/s41467-024-48911-z -
International Journal of Biological... Jun 2024Mre11 is one of important proteins that are involved in DNA repair and recombination by processing DNA ends to produce 3'-single stranded DNA, thus providing a platform...
Mre11 is one of important proteins that are involved in DNA repair and recombination by processing DNA ends to produce 3'-single stranded DNA, thus providing a platform for other DNA repair and recombination proteins. In this work, we characterized the Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-Mre11) biochemically and dissected the roles of its four conserved residues, which is the first report on Mre11 proteins from Thermococcus. Tba-Mre11 possesses exonuclease activity for degrading ssDNA and dsDNA in the 5'-3' direction, which contrasts with other reported Mre11 homologs. Maximum degradation efficiency was observed with Mn at 80 °C and at pH 7.5-9.5. In addition to possessing 5'-3' exonuclease activity, Tba-Mre11 has endonuclease activity that nicks plasmid DNA and circular ssDNA. Mutational data show that residues D10, D51 and N86 in Tba-Mre11 are essential for DNA degradation since almost no activity was observed for the D10A, D51A and N86A mutants. By comparison, residue D44 in Tba-Mre11 is not responsible for DNA degradation since the D44A mutant possessed the similar WT protein activity. Notably, the D44A mutant almost completely abolished the ability to bind DNA, suggesting that residue D44 is essential for binding DNA.
Topics: Thermococcus; Archaeal Proteins; DNA, Single-Stranded; Exodeoxyribonucleases; Amino Acid Sequence; Endonucleases; Mutation; Endodeoxyribonucleases
PubMed: 38810854
DOI: 10.1016/j.ijbiomac.2024.132654 -
Portable electrochemical aptasensor for highly sensitive detection of 3,3',4,4'-tetrachlorobiphenyl.Biosensors & Bioelectronics Sep 2024Aptamer-based electrochemical sensors are frequently used as independent, surface-functionalized, passive electrodes. However, their sensitivity and detection limits...
Aptamer-based electrochemical sensors are frequently used as independent, surface-functionalized, passive electrodes. However, their sensitivity and detection limits become limited, particularly when the electrode area is reduced to facilitate miniaturization. A mobile phone-based microfluidic electrochemical aptamer sensing platform for 3,3',4,4'-tetrachlorobiphenyl (PCB77) detection was developed in this work. This aptamer sensor utilized Exonuclease I (Exo I) and DNA/AuNPs/horseradish peroxidase (DNA/AuNPs/HRP) nanoprobes as a merged signal amplification method, which resulted in an increase in the electrochemical sensing performance. Sensitive detection of PCB77 was accomplished by functionalizing the hierarchically structured Au@MoS/CNTs/GO modified working/sensing electrode with the specific aptamer. The aptamer sensor was tested with different concentrations of PCB77 within the microfluidic platform. Afterward, the differential pulse voltammograms were recorded using a wireless integrated circuit device. Subsequently, the collected data was transmitted to a smartphone using Bluetooth communication. A detection limit of 0.0085 ng/L was obtained for PCB77 detection, with a detection range from 0.1 to 1000 ng/L. In addition, the detection of PCB77 in spiked water samples validated the possibility of using this aptamer sensor in a real environment, and the aptamer sensor demonstrated high selectivity in distinguishing PCB77 from other potential interfering species. The merging of electrochemical aptamer sensors with purposefully engineered microfluidic and integrated devices in this study is a novel and promising method that provides a dependable platform for on-site applications.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Polychlorinated Biphenyls; Gold; Electrochemical Techniques; Limit of Detection; Metal Nanoparticles; Exodeoxyribonucleases; Horseradish Peroxidase; Nanotubes, Carbon; Molybdenum; Equipment Design; Water Pollutants, Chemical; DNA; Smartphone
PubMed: 38810414
DOI: 10.1016/j.bios.2024.116434 -
Microbiology Spectrum Jul 2024, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. causes economic losses to livestock production and represents a...
UNLABELLED
, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect . The lowest limit of detection of the assay was 31 copies/μL for the gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of , showing promise for on-site surveillance.
IMPORTANCE
is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of are distributed worldwide, and cats and dogs are common hosts of . Therefore, rapid diagnosis of early infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of without false-negative results. Moreover, we detected the prevalence of in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of and could be applied in prevalence surveys of in stray dogs and cats.
Topics: Cats; Animals; Dogs; Toxoplasma; Dog Diseases; Cat Diseases; China; Toxoplasmosis, Animal; CRISPR-Cas Systems; Mice; Sensitivity and Specificity; CRISPR-Associated Proteins; Nucleic Acid Amplification Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; Molecular Diagnostic Techniques; Bacterial Proteins; Endodeoxyribonucleases
PubMed: 38809001
DOI: 10.1128/spectrum.03998-23 -
Biosensors & Bioelectronics Sep 2024To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro...
To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro transcripts (RNA), we developed a fluorescent biosensor named PDTCTR (PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter). This innovative biosensor integrates Recombinase Polymerase Amplification (RPA) with the Cas12f_ge4.1 system, facilitating the direct detection of double-stranded DNA (dsDNA). PDTCTR represents a significant leap forward, exhibiting a detection sensitivity that is a hundredfold greater than the original Cas12f1 system. It demonstrates the capability to detect Mycoplasma pneumoniae (M. pneumoniae) and Hepatitis B virus (HBV) with excellent sensitivity of 10 copies per microliter (16.8 aM) and distinguishes single nucleotide variations (SNVs) with high precision, including the EGFR (L858R) mutations prevalent in non-small cell lung cancer (NSCLC). Clinical evaluations of PDTCTR have demonstrated its high sensitivity and specificity, with rates ranging from 93%-100% and 100%, respectively, highlighting its potential to revolutionize diagnostic approaches for infectious diseases and cancer-related SNVs.This research underscores the substantial advancements in CRISPR technology for clinical diagnostics and its promising future in early disease detection and personalized medicine.
Topics: Biosensing Techniques; Humans; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Hepatitis B virus; DNA; Mycoplasma pneumoniae; CRISPR-Associated Proteins; Carcinoma, Non-Small-Cell Lung; Bacterial Proteins; Lung Neoplasms; Endodeoxyribonucleases; Pneumonia, Mycoplasma
PubMed: 38805891
DOI: 10.1016/j.bios.2024.116428 -
Nanoscale Jun 2024The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the...
The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.
Topics: Biosensing Techniques; Nanocomposites; Electrochemical Techniques; CRISPR-Cas Systems; Humans; CRISPR-Associated Proteins; Limit of Detection; Bacterial Proteins; Animals; Endodeoxyribonucleases; Biofouling
PubMed: 38804270
DOI: 10.1039/d4nr01618a -
Aging May 2024Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that...
Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.
Topics: Animals; Male; Female; Werner Syndrome; Liver; Mice; Proteomics; Aging; Werner Syndrome Helicase; Sex Characteristics; Disease Models, Animal
PubMed: 38795389
DOI: 10.18632/aging.205866