In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria.

In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.

Multiple analyses of main flavor components in reconstituted tobacco and transfer behavior of their key substances during heating.

Reconstituted tobacco (RT) is a product made by reprocessing tobacco waste, experiencing a growing demand for heat-not-burn products. The purpose of this study is to analyze the main flavor ingredients in RT aerosol, as well as the transfer behavior of key flavor substances from substrates to aerosol and the concentrations of these compounds in the substrate after heating. First, we demonstrated that the odor of four RT aerosol samples could be distinguished using an electronic nose. Through non-targeted analysis, 93 volatile compounds were detected by gas chromatography-mass spectrometry, and 286 non/semi-volatile compounds were identified by ultra-high-performance liquid electrophoresis chromatography-mass spectrometry in aerosol. Furthermore, we found that the formation of RT aerosol involves primarily evaporation and distillation, however, the total content delivered from unheated RT samples to aerosol remains relatively low due to compound volatility and cigarette filtration. Thermal reactions during heating indicated the pyrolysis of chlorogenic acid to generate catechol and resorcinol, while Maillard reactions involving glucose and proline produced 2,3-dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4-one. The study highlighted that heating RT at approximately 300°C could mitigate the production of harmful substances while still providing a familiar sensory experience with combusted tobacco.

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System.

Fungicide abuse leads to the emergence of fungicide-resistant fungal pathogens, thus posing a threat to agriculture and food safety. Here, we developed an isothermal amplification refractory mutation system (termed iARMS) allowing us to resolve genetic mutations, enabling rapid, sensitive, and potentially field-applicable detection of fungicide-resistant crop fungal pathogens. iARMS yielded a limit of detection of 25 aM via a cascade signal amplification strategy of recombinase polymerase amplification (RPA) and Cas12a-mediated collateral cleavage at 37 °C within 40 min. Specificity for fungicide-resistant Puccinia striiformis (P. striiformis) detection was guaranteed by RPA primers and the flexible sequence of gRNA. The iARMS assay allowed us to detect as low as 0.1% cyp51-mutated P. striiformis that showed resistance to the demethylase inhibitor (DMI), which was 50 times more sensitive than the sequencing techniques. Thus, it is promising for the discovery of rare fungicide-resistant isolates. We applied iARMS to investigate the emergence of fungicide-resistant P. striiformis in western China and found that its proportion was over 50% in Qinghai, Sichuan, and Xinjiang Province. iARMS can serve as a molecular diagnostic tool for crop diseases and facilitate precision plant disease management.

Wash-Free and High-Contrast Imaging of Single-Nucleotide Variation in Single Cells Using Transcription-Amplified Light-Up RNA Aptamer.

Single-nucleotide variation (SNV) imaging can indicate cellular heterogeneity and spatial pattern, but it remains challenging to produce high-gain signal while also yielding single-nucleotide resolution. Herein, we developed a light-up strategy for visualizing SNVs based on transcription amplification, enabling wash-free and high-contrast imaging of SNVs inside cells. The discrimination of SNVs is achieved by ligase-assisted transcription reaction. Employing a light-up RNA aptamer as a reporter eliminates nonspecific probe binding and the washing process and contributes to a 2-fold improvement of signal gain compared to that using the fluorescence in situ hybridization (FISH) method. The method allowed us to precisely quantify drug-resistant strains in the bacteria mixture and identify drug-resistant Salmonella enterica (S. enterica) isolated from poultry farm. Using this approach, we explored the colonization features of drug-resistant and drug-sensitive S. enterica in the mice intestinal tract and screened the prebiotics for Salmonella colonization inhibition. The SNV imaging method promises for the interrogation of genotypes in physiological and pathological states at the single-cell level.

CRISPR-based nucleic acid diagnostics for pathogens.

Pathogenic infection remains the primary threat to human health, such as the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutated variants, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we firstly describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges involved in on-site diagnostic assays and present emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.

Antibiotic resistance mechanism and diagnosis of common foodborne pathogens based on genotypic and phenotypic biomarkers.

The emergence of antibiotic-resistant bacteria due to the overuse or inappropriate use of antibiotics has become a significant public health concern. The agri-food chain, which serves as a vital link between the environment, food, and human, contributes to the large-scale dissemination of antibiotic resistance, posing a concern to both food safety and human health. Identification and evaluation of antibiotic resistance of foodborne bacteria is a crucial priority to avoid antibiotic abuse and ensure food safety. However, the conventional approach for detecting antibiotic resistance heavily relies on culture-based methods, which are laborious and time-consuming. Therefore, there is an urgent need to develop accurate and rapid tools for diagnosing antibiotic resistance in foodborne pathogens. This review aims to provide an overview of the mechanisms of antibiotic resistance at both phenotypic and genetic levels, with a focus on identifying potential biomarkers for diagnosing antibiotic resistance in foodborne pathogens. Furthermore, an overview of advances in the strategies based on the potential biomarkers (antibiotic resistance genes, antibiotic resistance-associated mutations, antibiotic resistance phenotypes) for antibiotic resistance analysis of foodborne pathogens is systematically exhibited. This work aims to provide guidance for the advancement of efficient and accurate diagnostic techniques for antibiotic resistance analysis in the food industry.

Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice.

Lead contamination in the environment tends to enter the food chain and further into the human body, causing serious health issues. Herein, we proposed a Csm6-DNAzyme tandem assay (termed cDNAzyme) using CRISPR/Cas III-A Csm6 and GR-5 DNAzyme, enabling one-pot and sensitive detection of lead contamination. We found that Pb2+-activated GR-5 DNAzyme produced cleaved substrates that can serve as the activator of Csm6, and the Csm6-DNAzyme tandem improved the sensitivity for detecting Pb2+ by 6.1 times compared to the original GR-5 DNAzyme. Due to the high specificity of DNAzyme, the cDNAzyme assay can discriminate Pb2+ from other bivalent and trivalent interfering ions and allowed precise detection of Pb2+ in water and food samples. Particularly, the assay can achieve one-step, mix-and-read detection of Pb2+ at room temperature. We used the cDNAzyme assay to investigate the accumulation of lead in mice, and found that lead accumulated at higher levels in the colon and kidney compared to the liver, and most of the lead was excreted. The cDNAzyme assay is promising to serve as analytical tools for lead-associated environmental and biosafety issues.

CRISPR-Cas based molecular diagnostics for foodborne pathogens.

Foodborne pathogenic infection has brought multifaceted issues to human life, leading to an urgent demand for advanced detection technologies. CRISPR/Cas-based biosensors have the potential to address various challenges that exist in conventional assays such as insensitivity, long turnaround time and complex pretreatments. In this perspective, we review the relevant strategies of CRISPR/Cas-assisted diagnostics on foodborne pathogens, focusing on biosensing platforms for foodborne pathogens based on fluorescence, colorimetric, (electro)chemiluminescence, electrochemical, and surface-enhanced Raman scattering detection. It summarizes their detection principles by the clarification of foodborne pathogenic bacteria, fungi, and viruses. Finally, we discuss the current challenges or technical barriers of these methods against broad application, and put forward alternative solutions to improve CRISPR/Cas potential for food safety.

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.

CRISPR/Cas14a-Based Isothermal Amplification for Profiling Plant MicroRNAs.

MicroRNAs (miRNAs) play key roles in biological processes in plants, such as stress resistance, yet can hardly be quantified by an enzyme-involved terminal polymerization process due to their 2'-O-methyl modifications at the 3'-terminal. Herein, we proposed a CRISPR/Cas14a-based rolling circle amplification (termed Cas14R) assay, allowing reverse transcription-free and demethylation-free detection of plant miRNAs with single-nucleotide resolution. The employment of target-templated rolling circle amplification circumvents the extension of the unaccessible 2'-O-methyl group at the 3'-terminal. Particularly, the activated Cas14a confers the trans-cleavage activity for identifying target single-stranded DNA sequences without the necessity of the protospacer adjacent motif, generalizing the detection of miRNA sequences and the integration of different isothermal amplification techniques. Ultimately, the Cas14R assay has been applied to profile miR156a to evaluate the ripeness process of banana, indicating its feasibility in analyzing the roles of miRNAs in biological processes of plants.

Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global health emergency, and its gene mutation and evolution further posed uncertainty of epidemic risk. Herein, we reported a light-up CRISPR-Cas13 transcription amplification method, which enables the detection of SARS-CoV-2 and its mutated variants. Sequence specificity was ensured by both the ligation process and Cas13a/crRNA recognition, allowing us to identify viral RNA mutation. Light-up RNA aptamer allows sensitive output of amplification signals via target-activated ribonuclease activity of CRISPR-Cas13a. The RNA virus assay has been designed to detect coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS), and SARS, as well as the influenza viruses such as, H1N1, H7N9, and H9N2. It was accommodated to sense as low as 82 copies of SARS-CoV-2. Particularly, it allowed us to strictly discriminate key mutation of the SARS-CoV-2 variant, D614G, which may induce higher epidemic and pathogenetic risk. The proposed RNA virus assays are promising for point-of-care monitoring of SARS-CoV-2 and its risking variants.

Lighting up single-nucleotide variation in situ in single cells and tissues.

The ability to 'see' genetic information directly in single cells can provide invaluable insights into complex biological systems. In this review, we discuss recent advances of in situ imaging technologies for visualizing the subtlest sequence alteration, single-nucleotide variation (SNV), at single-cell level. The mechanism of recently developed methods for SNV discrimination are summarized in detail. With recent developments, single-cell SNV imaging methods have opened a new door for studying the heterogenous and stochastic genetic information in individual cells. Furthermore, SNV imaging can be used on morphologically preserved tissue, which can provide information on histological context for gene expression profiling in basic research and genetic diagnosis. Moreover, the ability to visualize SNVs in situ can be further developed into in situ sequencing technology. We expect this review to inspire more research work into in situ SNV imaging technologies for investigating cellular phenotypes and gene regulation at single-nucleotide resolution, and developing new clinical and biomedical applications.

Single-Cell Imaging of m6 A Modified RNA Using m6 A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6 AISH-PLA).

N6 -methyladenosine (m6 A) modification-the most prevalent mammalian RNA internal modification-plays key regulatory roles in mRNA metabolism. Current approaches for m6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m6 A-specific in situ hybridization mediated proximity ligation assay (m6 AISH-PLA) for cellular imaging of m6 A RNA, allowing to identify m6 A modification at specific location in RNAs and image m6 A RNA with single-cell and single-molecule resolution. Using m6 AISH-PLA, we investigated the m6 A level and subcellular location of HSP70 RNA103-m6 A in response to heat shock stress, and found an increased m6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6 AISH-PLA can serve in the study of m6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.

Highly Sensitive MicroRNA Detection by Coupling Nicking-Enhanced Rolling Circle Amplification with MoS2 Quantum Dots.

In this work, a label-free and highly sensitive fluorescence assay was constructed for microRNA detection. Nicking-enhanced rolling circle amplification (RCA) induced by G-quadruplex formation is coupled with inner filter effect (IFE)-based quenching effects of MoS2 quantum dots (MoS2 QDs). The padlock probe contains a recognition sequence to target microRNA and an accessible nicking site. The padlock probe is cyclized upon hybridization with target microRNA. Sequentially, amplification initiates a production of a long-concatenated sequence of circular probes. Abundant G-quadruplex sequences are produced via the nicking process and then used as the trigger to initiate the next RCA. In the presence of hemin, numerous hemin/G-quadruplex DNAzymes are formed, which catalyze the oxidation of o-phenylenediamine (OPD) into the colored product 2,3-diaminophenazine, resulting in quenching of the fluorescence of MoS2 QDs. This sensing strategy enables detection of microRNA let-7a with high selectivity and a detection limit of 4.6 fM. The as-prepared sensor was applied for detecting microRNA let-7a in dilute human serum samples and achieved a satisfactory recovery rate, demonstrating its potential in clinic diagnosis of microRNA-associated disease and biochemical research.

Graphene/aptamer probes for small molecule detection: from in vitro test to in situ imaging.

Small molecules are key targets in molecular biology, environmental issues, medicine and food industry. However, small molecules are challenging to be detected due to the difficulty of their recognition, especially in complex samples, such as in situ in cells or animals. The emergence of graphene/aptamer probes offers an excellent opportunity for small molecule quantification owing to their appealing attributes such as high selectivity, sensitivity, and low cost, as well as the potential for probing small molecules in living cells or animals. This paper (with 130 refs.) will review the application of graphene/aptamer probes for small molecule detection. We present the recent progress in the design and development of graphene/aptamer probes enabling highly specific, sensitive and rapid detection of small molecules. Emphasis is placed on the success in their development and application for monitoring small molecules in living cells and in vivo systems. By discussing the key advances in this field, we wish to inspire more research work of the development of graphene/aptamer probes for both on-site or in situ detection of small molecules and its applications for investigating the functions of small molecules in cells in a dynamic way. Graphical abstract Graphene/aptamer probes can be used to construct different platforms for detecting small molecules with high specificity and sensitivity, both in vitro and in situ in living cells and animals.

Cascade Transcription Amplification of RNA Aptamer for Ultrasensitive MicroRNA Detection.

MicroRNAs (miRNAs) play a critical role in multifarious biological processes and being deemed to be important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Thus, assays for sensitive and accurate quantification of miRNAs are highly demanded. Herein, we have constructed a RNA aptamer involved cascade transcription amplification method (termed RACTA), enabling label-free, ultrasensitive, and specific detection of miRNA. Target miRNA-initiated strand-displacement amplification would allow for the production of plenty of ssDNA that triggers the subsequent transcriptional amplification of spinach RNA aptamers. Consequently, transcribed tremendous spinach aptamers activated fluorophore DFHBI (( Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1 H-imidazol-5(4 H)-one) for miRNA quantitative analysis. RACTA outperforms conventional strand displacement amplification (SDA) at both background and amplification rate due to the light-up mechanism of DFHBI dye-Spinach aptamer and cascade signal amplification of RACTA. Thus, the signal-to-noise ratio of RACTA was increased by about 20-fold compared to that of SDA. This RACTA assay could confer a highly sensitive detection of miRNA with a detection limit of 5.12 × 10-18 M and excellent specificity enabling differentiation between miRNAs and homologous families. Besides, this assay has been successfully demonstrated for quantification of miRNAs in different cell lines. Therefore, the proposed method holds great potential for miRNA biomarker based early diagnosis and prognosis monitoring.

RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing.

CRISPR/Cas9 has already become a powerful tool for genomic manipulation, and further engineering of the system allows it to be precisely regulated in response to external signals, thus, broadening its application possibilities, such as biosensing or bioimaging. However, most stimuli-responsive CRISPR systems are built based on elaborately designed and engineered inducible Cas9 proteins, and external stimuli are still mostly limited as small molecules and light. To construct more precise and easy-to-build responsive CRISPR systems and broaden their responsive species, we seek to engineer conditional guide RNA, rather than Cas9 protein, to mediate conditional CRISPR corresponding to logic operation. Here, we construct mRNA-sensing CRISPR by gRNA reconfiguration and toehold mediated strand displacement, in which each target site could be independently controlled. We show that switches can be embedded into the gRNA and used as RNA sensors, capable of detecting multiple mRNA inputs orthogonally and providing CRISPR/Cas9 response outputs. NOR and NAND logical gates are also constructed, demonstrating its orthogonality and programmability. This strategy promises potential uses in constructing genetic circuits to detect endogenous mRNAs and initiate cellular responses.

Trypsin-Amplified Aerolysin Nanopore Amplified Sandwich Assay for Attomolar Nucleic Acid and Single Bacteria Detection.

Nanopore technology is promising for the next-generation of nucleic acid-based diagnosis. However, sequence reservation could still be hardly achieved in low-concentration. Herein, we propose a trypsin-activated catalysis reaction for amplified detection, which substantially improves the sensitivity of nanopore technique. The proposed trypsin-amplified nanopore amplified sandwich assay (tNASA) could contribute to a sensitivity approximately 100 000 times higher based on nucleic acid probe design. Remarkably, tNASA is capable of attomolar nucleic acid and single cell detection by using a miniaturized current amplifier without alignment algorithm. Also it allows 10 pathogenic species in serum to be accurately and robustly profiled, thus be utilized for the diagnosis of infectious diseases. tNASA may evolve the construction of nanopore techniques for nucleic acid detection and would facilitate its translation for pocket diagnosis and precision medicine.

Emerging Applications of Nanotechnology for Controlling Cell-Surface Receptor Clustering.

The spatial organization of cell-surface receptors plays an important role in defining cell fate. Recently, the development of strategies for the direct regulation of receptor clustering using nanomaterials has aroused enormous interest. In this review, we discuss the mechanisms and features of recently developed nanomaterial-based strategies to control the nanoscale distribution of cell binding ligands and regulate cell behavior. We expect this review to inspire innovative work on manipulating cell functions by controlling the clustering of cell surface receptors.

Direct Visualization of Single-Nucleotide Variation in mtDNA Using a CRISPR/Cas9-Mediated Proximity Ligation Assay.

The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging-associated diseases. Imaging of single-nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9-mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single-molecule resolution. Using CasPLA, we observed a mtDNA-transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy genomic loci ( KRAS gene) in the nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.