In-Field Molecular Diagnostics of Plant Pathogens Using Bioluminescent CRISPR-Guided Caspase Assay.

In-field molecular diagnostics of plant pathogens are critical for crop disease management and precision agriculture, but tools are still lacking. Herein, we present a bioluminescent molecular diagnostic assay capable of detecting viable pathogens directly in minimally processed plant samples, enabling rapid and precise in-field crop disease diagnosis. The assay, called bioluminescent craspase diagnostics (BioCrastics), leverages newly discovered RNA-activated protease of CRISPR (Craspase) with enzymatic luminescence to generate a cascaded amplification, thus bypasses nucleic acid purification and amplification while achieving sub-nanogram sensitivity for fungal pathogens.

Machine learning assisted CRCA multi-color fluorescent droplet imaging for high-sensitivity simultaneous detection of Maillard hazards.

Acrylamide and advanced glycation end products are two typical hazards produced simultaneously through the Maillard reaction of food. Traditional instrumental detection methods require specialized instrument operation, resulting in low detection efficiency. Here, a red-green dual fluorescence detection system combined with Nt.

High-Fidelity Profiling of Multiple Nearby Mutations via Cooperative Recognition.

Detection methods with single-nucleotide specificity are essential tools for nucleic acid analysis in diverse clinical and biological settings. However, both hybridization-based and enzyme-based methods are only effective for discriminating single-nucleotide mutations at certain positions, making it difficult to detect nucleic acid targets having multiple nearby mutations. Herein, we describe the design of cooperative recognition probes (CRPs) that integrate both hybridization and ligation-based recognition mechanisms and thus are highly effective for discriminating mutations throughout all positions.

One-pot enzyme-free amplified detection of trace mercury contamination in aquatic products using mid-toehold-mediated-DNA Strand displacement.

Mercury pollution in aquatic products poses critical risks due to bioaccumulation and biomagnification in the food chain, necessitating tools for rapid, sensitive and accurate detection of mercury in food samples. Herein, we developed a mix-and-read, enzyme-free amplified method for one-pot detection of mercury pollution in aquatic products based on mid-toehold mediated- DNA strand displacement reaction. The key innovation of the method lies in the design of a pre-quenched T-rich duplex probe that enables Hg-activated strand displacement through T-Hg-T coordination, releasing two fluorophores per binding event to achieve intrinsic signal amplification.

Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay.

N6-Methyladenosine (mA) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRmA assay) allowing for preamplification-free, sensitive, and rapid detection of RNA mA modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify mA RNA with single-base resolution.

Imaging Low-Abundance, Short Cellular RNAs Using G-quadruplex FISH.

Visualizing cellular RNAs enables spatial resolution at the single-cell level. Fluorescence hybridization (FISH) is a facile tool; however, it is not sensitive enough to detect low-abundance or short RNAs in cells due to limited signal gain. Here, we present G-quadruplex FISH, a sensitive RNA imaging method that synergizes G-quadruplex peroxidase-driven tyramide deposition with proximity labeling chemistry.

Preamplification-Free Detection of Viable Microorganisms in Fermentation Using Tandem CRISPR Nuclease Probe.

Accurate detection of viable bacteria is crucial for evaluating and monitoring the fermentation process. However, the complexity of fermentation samples presents challenges to developing precise and rapid detection tools. Here, we present a Cas13a-Csm6 tandem nuclease probe capable of the one-pot detection of viable microorganisms during fermentation, eliminating the need for nucleic acid preamplification.

Simultaneous Monitoring of Tyrosinase and ATP in Thick Brain Tissues Using a Single Two-Photon Fluorescent Probe.

Cellular redox homeostasis and energy metabolism in the central nervous system are associated with neurodegenerative diseases. However, their real-time and concurrent monitoring in thick tissues remains challenging. Herein, a single dual-emission two-photon fluorescent probe (named DST) is designed for the simultaneous tracking of tyrosinase (TYR) and adenosine triphosphate (ATP), thereby enabling the real-time monitoring of both neurocellular redox homeostasis and energy metabolism in brain tissue.

Accurate Prediction of CRISPR/Cas13a Guide Activity Using Feature Selection and Deep Learning.

CRISPR/Cas13a serves as a key tool for nucleic acid tests; therefore, accurate prediction of its activity is essential for creating robust and sensitive diagnosis. In this study, we create a dual-branch neural network model that achieves high prediction accuracy and classification performance across two independent CRISPR/Cas13a data sets, outperforming previously published models relying solely on sequence features. The model integrates direct sequence encoding with descriptive features and yields 99 key descriptive features out of 1553, extracted through statistical analysis, which critically influence guide-target interactions and Cas13a guide activity.

Argonaute-mediated dual recognition enables the detection of trace single-nucleotide-mutated fungicide-resistant fungal pathogens.

Detection of low-abundance mutations for the early discovery of fungicide-resistant fungal pathogens is highly demanded, but remains challenging. Herein, we developed a dual-recognition strategy, termed PARPA, involving s Argonaute (pfAgo)-mediated elimination of wild-type fungal genes and CRISPR/Cas12a-based amplicon recognition. This assay can detect fungicide-resistant at relative abundances as low as 0.

Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.

Background: The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge.

Methods: Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification.

Dynamic, Single-cell Monitoring of RNA Modifications Response to Viral Infection Using a Genetically Encoded Live-cell RNA Methylation Sensor.

RNA modifications, such as N6-methylation of adenosine (mA), serve as key regulators of cellular behaviors, and are highly dynamic; however, tools for dynamic monitoring of RNA modifications in live cells are lacking. Here, we develop a genetically encoded live-cell RNA methylation sensor that can dynamically monitor RNA mA level at single-cell resolution. The sensor senses RNA mA in cells via affinity-induced cytoplasmic retention using a nuclear location sequence-fused mA reader.

Correction: DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination.

Correction for 'DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination' by Ruijie Deng , , 2024, , 5976-5979, https://doi.org/10.1039/D4CC01852D.

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria.

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica).

High-contrast imaging of cellular non-repetitive drug-resistant genes dead Cas12a-labeled PCR.

imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes.

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.

Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification.

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one.

Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test.

The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution.

Multiplexing Imaging of Closely Located Single-Nucleotide Mutations in Single Cells via Encoded PCR.

Mutation accumulation in RNAs results in closely located single-nucleotide mutations (SNMs), which is highly associated with the drug resistance of pathogens. Imaging of SNMs in single cells has significance for understanding the heterogeneity of RNAs that are related to drug resistance, but the direct "see" closely located SNMs remains challenging. Herein, we designed an encoded ligation-mediated polymerase chain reaction method (termed enPCR), which enabled the visualization of multiple closely located SNMs in bacterial RNAs.

Single-Cell Imaging of mRNA by Target RNA-Initiated RCA.

We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination.

Hazardous lead ions (Pb) even at a minute level can pose side effects on human health, highlighting the need for tools for trace Pb detection. Herein, we present a DNAzyme-activated CRISPR assay (termed DzCas12T) for sensitive and one-pot detection of lead contamination. Using an extension-bridged strategy eliminates the need for separation to couple the DNAzyme recognition and CRISPR reporting processes.

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

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 hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs.

In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor.

Mutations in mitochondrial DNA (mtDNA) play critical roles in many human diseases. In vivo visualization of cells bearing mtDNA mutations is important for resolving the complexity of these diseases, which remains challenging. Here we develop an integrated nano Cas12a sensor (InCasor) and show its utility for efficient imaging of mtDNA mutations in live cells and tumor-bearing mouse models.

Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels.