Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing.

mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution.

In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community.

Ferrobotic swarms enable accessible and adaptable automated viral testing.

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

Fast and sensitive CRISPR detection by minimized interference of target amplification.

Despite the great potential of CRISPR-based detection, it has not been competitive with other market diagnostics for on-site and in-home testing. Here we dissect the rate-limiting factors that undermine the performance of Cas12b- and Cas13a-mediated detection. In one-pot testing, Cas12b interferes with loop-mediated isothermal amplification by binding to and cleaving the amplicon, while Cas13a directly degrades the viral RNA, reducing its amplification. We found that the protospacer-adjacent motif-interacting domain engineered Cas12b accelerated one-pot testing with 10-10,000-fold improved sensitivity, and detected 85 out of 85 SARS-CoV-2 clinical samples with a sensitivity of 0.5 cp µl-1, making it superior to wild-type Cas12b. In parallel, by diminishing the interference of Cas13a with viral RNA, the optimized Cas13a-based assay detected 86 out of 87 SARS-CoV-2 clinical samples at room temperature in 30 min with a sensitivity of 0.5 cp µl-1. The relaxed reaction conditions and improved performance of CRISPR-based assays make them competitive for widespread use in pathogen detection.

An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing.

CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.

Testing for SARS-CoV-2: lessons learned and current use cases.

SUMMARYThe emergence and worldwide dissemination of SARS-CoV-2 required both urgent development of new diagnostic tests and expansion of diagnostic testing capacity on an unprecedented scale. The rapid evolution of technologies that allowed testing to move out of traditional laboratories and into point-of-care testing centers and the home transformed the diagnostic landscape. Four years later, with the end of the formal public health emergency but continued global circulation of the virus, it is important to take a fresh look at available SARS-CoV-2 testing technologies and consider how they should be used going forward. This review considers current use case scenarios for SARS-CoV-2 antigen, nucleic acid amplification, and immunologic tests, incorporating the latest evidence for analytical/clinical performance characteristics and advantages/limitations for each test type to inform current debates about how tests should or should not be used.

Parasite-derived microRNA let-7-5p detection for metacestodiasis based on rolling circular amplification-assisted CRISPR/Cas9.

Metacestodiasis is an infectious disease caused by the larval stage of cestode parasites. This disease poses a serious health hazard to wildlife, livestock, and humans, and it incurs substantial economic losses by impacting the safety of the livestock industry, the quality of meat production, and public health security. Unfortunately, there is currently no available molecular diagnostic method capable of distinguishing cysticercus- and Echinococcus-derived microRNAs (miRNAs) from other helminthes and hosts in the plasma of metacestode-infected animals. This study aims to develop a specific, sensitive, and cost-efficient molecular diagnostic method for cysticercosis and echinococcosis, particularly for early detection. The study developed a rolling circular amplification (RCA)-assisted CRISPR/Cas9 detection method based on parasite-derived miRNA let-7-5p. Using a series of dilutions of the let-7 standard, the limit of detection (LOD) of the qPCR, RCA, and RCA-assisted CRISPR/Cas9 methods was compared. The specificity of qPCR and CRISPR/Cas9 was evaluated using four artificially synthesized let-7 standards from different species. A total of 151 plasma samples were used to evaluate the diagnostic performance. Additionally, the study also assessed the correlation between plasma levels of let-7-5p, the number of Taenia pisiformis cysticerci, and the weight of Echinococcus multilocularis cysts. The results demonstrated that the RCA-assisted CRISPR/Cas9 assay could significantly distinguish let-7 from cestodes and other species, achieving a LOD of 10 aM; the diagnostic sensitivity and specificity for rabbit cysticercosis and mouse E. multilocularis were 100% and 97.67%, and 100% and 100%, respectively. Notably, let-7-5p gradually increased in the plasma of T. pisiformis-infected rabbits from 15 days post infection (dpi), peaked at 60 dpi, and persisted until 120 dpi. In E. multilocularis-infected mice, let-7-5p gradually increased from 15 dpi and persisted until 90 dpi. Furthermore, the expression of let-7-5p positively correlated with the number of cysticerci and cyst weight. These results indicated that the let-7-5p-based RCA-assisted CRISPR/Cas9 assay is a sensitive and specific detection method that can be used as a universal diagnostic method for metacestodiasis, particularly for early diagnosis (15 dpi).

Self-coalescing flows in microfluidics for pulse-shaped delivery of reagents.

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.

Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging.

Spatial visualization of single-cell transcripts is limited by signal specificity and multiplexing. Here, we report hierarchical DNA branch assembly-encoded fluorescent nanoladders, which achieve denoised and highly multiplexed signal amplification for single-molecule transcript imaging. This method first offers independent RNA-primed rolling circle amplification without nonspecific amplification based on circular DNAzyme. It then executes programmable DNA branch assembly on these amplicons to encode virtual signals for visualizing numbers of targets by FISH. In theory, more virtual signals can be encoded via the increase of detection spectral channels and repeats of the same sequences on barcode. Our method almost eliminates nonspecific amplification in fixed cells (reducing nonspecific spots of single cells from 16 to nearly zero), and achieves simultaneous quantitation of nine transcripts by using only two detection spectral channels. We demonstrate accurate RNA profiling in different cancer cells, and reveal diverse localization patterns for spatial regulation of transcripts.

Rapid in situ RNA imaging based on Cas12a thrusting strand displacement reaction.

RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.

Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Chlamydia trachomatis Seroassays Used in Epidemiologic Research: A Narrative Review and Practical Considerations.

Chlamydia trachomatis (CT) is a sexually transmitted infection that can lead to adverse reproductive health outcomes. CT prevalence estimates are primarily derived from screening using nucleic acid amplification tests (NAATs). However, screening guidelines in the United States only include particular subpopulations, and NAATs only detect current infections. In contrast, seroassays identify past CT infections, which is important for understanding the public health impacts of CT, including pelvic inflammatory disease and tubal factor infertility. Older seroassays have been plagued by low sensitivity and specificity and have not been validated using a consistent reference measure, making it challenging to compare studies, define the epidemiology of CT, and determine the effectiveness of control programs. Newer seroassays have better performance characteristics. This narrative review summarizes the "state of the science" for CT seroassays that have been applied in epidemiologic studies and provides practical considerations for interpreting the literature and employing seroassays in future research.

The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing (December 2023).

Accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (COVID-19) and for identifying asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The number of available SARS-CoV-2 nucleic acid detection tests continues to increase as does the COVID-19 diagnostic literature. Thus, the Infectious Diseases Society of America (IDSA) developed an evidence-based diagnostic guideline to assist clinicians, clinical laboratorians, patients, and policymakers in decisions related to the optimal use of SARS-CoV-2 nucleic acid amplification tests. In addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss nuances of test result interpretation in a variety of practice settings, and highlight important unmet research needs related to COVID-19 diagnostic testing. IDSA convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of SARS-CoV-2 molecular diagnostics. Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to assess the certainty of evidence and make testing recommendations. The panel agreed on 12 diagnostic recommendations. Access to accurate SARS-CoV-2 nucleic acid testing is critical for patient care, hospital infection prevention, and the public health response to COVID-19 infection. Information on the clinical performance of available tests continues to grow, but the quality of evidence of the current literature to support this updated molecular diagnostic guideline remains moderate to very low. Recognizing these limitations, the IDSA panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having COVID-19. In addition, testing is suggested for asymptomatic individuals with known or suspected contact with a COVID-19 case when the results will impact isolation/quarantine/personal protective equipment (PPE) usage decisions. Evidence in support of rapid testing and testing of upper respiratory specimens other than nasopharyngeal swabs, which offer logistical advantages, is sufficient to warrant conditional recommendations in favor of these approaches.

Hairpin-Empowered Invasive Reaction Combined with Catalytic Hairpin Assembly Cascade Amplification for the Specific Detection of Single-Nucleotide Polymorphisms.

Single-nucleotide polymorphism (SNP) is widely used in the study of disease-related genes and in the genetic study of animal and plant strains. Therefore, SNP detection is crucial for biomedical diagnosis and treatment as well as for molecular design breeding of animals and plants. In this regard, this article describes a novel technique for detecting SNP using flap endonuclease 1 (FEN 1) as a specific recognition element and catalytic hairpin assembly (CHA) cascade reaction as a signal amplification strategy. The mutant target (MT) was hybridized with a biotin-modified upstream probe and hairpin-type downstream probe (DP) to form a specific three-base overlapping structure. Then, FEN 1 was employed for three-base overlapping structure-specific recognition, namely, the precise SNP site identification and the 5' flap of DP dissociation. After dissociation, the hybridized probes were magnetically separated by a streptavidin-biotin complex. Especially, the ability to establish such a hairpin-type DP provided a powerful tool that could be used to hide the cut sequence (CS) and avoid false-positive signals. The cleaved CS initiated the CHA reaction and allowed superior fluorescence signal generation. Owing to the high specificity of FEN 1 for single base recognition, only the MT could be distinguished from the wild-type target and mismatched DNA. Owing to the dual signal amplification, as low as 0.36 fM MT and 1% mutation abundance from the mixtures could be detected, respectively. Furthermore, it could accurately identify SNPs from human cancer cells, as well as soybean leaf genome extracts. This strategy paves the way for the development of more precise and sensitive tools for diagnosing early onset diseases as well as molecular design breeding tools.

Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification.

Prokaryotic Argonautes (pAgos) have been recently used in many nucleic acid biosensing applications but have rarely been used for regulating the isothermal amplification system. Herein, we reported Thermus thermophilus Argonaute (TtAgo)-mediated background-suppressed exponential isothermal amplification (EXPAR) as the first example to explore the binding activity of pAgos toward regulation of the amplification template. It was demonstrated that thermophilic pAgos efficiently eliminated nonspecific hybridization between templates by their binding affinity with the template, resulting in greatly enhancing the specificity of EXPAR. TtAgo-mediated, background-suppressed EXPAR was employed to detect miRNA with a detection limit of 10-15 M, which was 1000 times and 100 times more sensitive than that of traditional RT-PCR and EXPAR, respectively. This method further showed good performance in discriminating cancer patients from healthy individuals, indicating its potential for practical clinical applications.

Toehold Strand Displacement-Mediated Exponential HCR for Highly Sensitive and Specific Analysis of miRNA in Living Cells.

As an important disease biomarker, the development of sensitive detection strategies for miRNA, especially intracellular miRNA imaging strategies, is helpful for early diagnosis of diseases, pathological research, and drug development. Hybridization chain reaction (HCR) is widely used for miRNA imaging analysis because of its high specificity and lack of biological enzymes. However, the classic HCR reaction exhibits linear amplification with low efficiency, limiting its use for the rapid analysis of trace miRNA in living cells. To address this problem, we proposed a toehold-mediated exponential HCR (TEHCR) to achieve highly sensitive and efficient imaging of miRNA in living cells using ß-FeOOH nanoparticles as transfection vectors. The detection limit of TEHCR was as low as 92.7 fM, which was 8.8 × 103 times lower compared to traditional HCR, and it can effectively distinguish single-base mismatch with high specificity. The TEHCR can also effectively distinguish the different expression levels of miRNA in cancer cells and normal cells. Furthermore, TEHCR can be used to construct OR logic gates for dual miRNA analysis without the need for additional probes, demonstrating high flexibility. This method is expected to play an important role in clinical miRNA-related disease diagnosis and drug development as well as to promote the development of logic gates.

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris.

Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay's efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/µL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102-103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.

Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 µL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.