linear DNA crispr

BACKGROUND: MicroRNA-21 has been determined to be the only microRNA overexpressed in 11 types of solid tumors, making it an excellent candidate as a biomarker for disease diagnosis and therapy. Photoelectrochemical (PEC) biosensors have been widely used for quantification of microRNA-21. However, most PEC biosensing processes still suffer from some problems, such as the difficulty of avoiding the influence of interferents in complex matrices and the false-positive signals. There is a pressing need for establishing a sensitive and stable PEC method to detect microRNA-21...

Inherited and germ-line de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA-origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate...

The sensitive and accurate detection of ochratoxin A (OTA) is crucial for public health due to its high toxicity. Herein, using Au nanoparticle (NP)-attached CdS/UiO-66-NH2 heterostructures as photoactive materials, a photoelectrochemical (PEC) aptasensor was presented for the ultrasensitive assay of OTA based on a competitive displacement reaction triggering the trans-cleavage ability of CRISPR/Cas12a. In this sensing strategy, methylene blue-labeled single-stranded DNA (MB-ssDNA) was immobilized on the Au NPs/CdS/UiO-66-NH2 electrode to accelerate the separation of the photogenerated carrier, thus producing a significantly increased PEC response...

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other...

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) system enables sensitive and specific detection of biomolecules, thanks to its programmability, high fidelity, and powerful signal amplification capabilities. Herein, a universal smartphone-assisted label-free G-quadruplex (G4) DNAzyme-based chemiluminescence CRISPR/Cas12a biosensing platform (G4CLCas) is firstly described that achieves on-site, ultrasensitive visual detection of nucleic acid and non-nucleic acid targets...

An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin...

The rapidly advanced CRISPR/Cas biosensing technology provides unprecedent potential for the development of novel biosensing systems. It provides a new approach for realizing rapid, sensitivity and highly specific pathogen nucleic acid detection, with the capability to combine other technologies, including Polymerase Chain Reaction or isothermal amplifications. The detection of Helicobacter pylori (H. pylori), one of the most common human pathogens to cause various gastroduodenal diseases, has also been explored with the assistance of CRISPR/Cas systems...

BACKGROUND: Major psychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BPD) are complex genetic mental illnesses. Their non-Mendelian features, such as those observed in monozygotic twins discordant for SCZ or BPD, are likely complicated by environmental modifiers of genetic effects. 5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark in gene regulation, and whether it is linked to genetic variants that contribute to non-Mendelian features remains largely unexplored...

BACKGROUND: Escherichia coli (E.coli) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract, posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors...

BACKGROUND: Salmonella infection severely threatens human health and causes substantial medical and financial concerns. Sensitive and specific detection of Salmonella in food samples is crucial but remains challenging. While some traditional assays for S. typhimurium are reliable, they suffer from various limitations, such as being time-consuming (culture-based methods), involving intricate nucleic molecular extraction (polymerization chain reaction, PCR), and exhibiting inadequate sensitivity (enzyme-linked immunosorbent assay, ELISA)...

We propose a CRISPR/Cas12a-mediated recombinase polymerase amplification (RPA) detection method that combines RPA with Cas12a cleavage for the detection of halal food adulteration, which is of global concern, particularly for Muslim consumers. We optimized the reagent concentrations for the Cas12a cleavage steps and designed and screened gRNA targeting a conserved area of the mitochondrial cytochrome C oxidase subunit I ( COX1 ) gene. This procedure successfully detected the presence of porcine components as low as 5 pg/μL in the linear range of 5-1000 pg/μL...

MicroRNA (miRNA) has gained significant attention as a potential biomarker for cancer clinics, and there is an urgent need for developing sensing strategies with high selectivity, sensitivity, and low background. In vitro diagnosis based on Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated protein (CRISPR/Cas) technology could simplify the detection procedure, improve sensitivity and selectivity, and has broad application prospects as the next-generation molecular diagnosis technology...

BACKGROUND: Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population-level trials. As a result, a proportion of patients are under- or over-dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy...

In this work, combined with the high amplification efficiency of hybridization chain reaction (HCR), high specificity of the CRISPR/Cas12a system, and convenience of the homogeneous electrochemiluminescence (ECL) assay based on the regulation of negative charge on the reporting probes, a sensitive ECL biosensor for hepatitis B virus DNA (chosen as a model target) had been developed. The initiator chain trigger DNA that can induce HCR amplification is modified on the surface of ruthenium bipyridine-doped silica nanoparticles (Ru@SiO2 NPs) first, and large amounts of negative charges modified on the particles were achieved through the HCR amplification reaction...

CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line...

The clustered regularly interspaced short palindromic repeats (CRISPR) system provides a new molecular diagnostic tool for construction of biosensor platforms due to its high programmability and target specificity. Herein, we developed a CRISPR-empowered electrochemical biosensor by combining the advantages of CRISPR/Cas13a and primer exchange reaction (PER), named PER-E-CRISPR, for target amplification-free and sensitive detection of miR-21. Dual-signal amplification procedures involve the binding of target miR-21 induced by CRISPR-based amplification, along with the hybridization of multiple short single-stranded DNA strands with PER concatemers...

Environmental DNA (eDNA) research holds great promise for improving biodiversity science and conservation efforts by enabling worldwide species censuses in near real-time. Current eDNA methods face challenges in detecting low-abundance ecologically important species. In this study, we used isothermal recombinase polymerase amplification (RPA)-CRISPR/Cas detection to test Ctenopharyngodon idella . RPA-CRISPR-Cas12a detected 6.0 eDNA copies/μL within 35 min. Ecologically rare species were identified in the Three Gorges Reservoir Area (TGRA) using functional distinctiveness and geographical restrictiveness, with seven fish species (9%) classified as potentially ecologically rare including three species in this investigation...

Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis , utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR...

Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans-cleavage of a reporter molecule...

CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates ( k cat ) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM)...