Cleavage Of Single-stranded DNA Research Articles

Dual electrochemical signal “signal-on-off” sensor based on CHA-Td-HCR and CRISPR-Cas12a for MUC1 detection

Mucin 1 (MUC1) is frequently overexpressed in various cancers and is essential for early cancer detection. Current methods to detect MUC1 are expensive, time-consuming, and require skilled personnel. Therefore, developing a simple, sensitive, highly selective MUC1 detection sensor is necessary. In this study, we proposed a novel “signal-on-off” strategy that, in the presence of MUC1, synergistically integrates catalytic hairpin assembly (CHA) with DNA tetrahedron (Td)-based nonlinear hybridization chain reaction (HCR) to enhance the immobilization of electrochemically active methylene blue (MB) on magnetic nanoparticles (MNP), marking the MB signal “on”. Concurrently, the activation of CRISPR-Cas12a by isothermal amplification products triggers the cleavage of single-stranded DNA (ssDNA) at the electrode surface, resulting in a reduction of MgAl-LDH@Fc-AuFe-MIL-101 (containing ferrocene, Fc) on the electrode, presenting the “signal-off” state. Both MB and MgAl-LDH@Fc-AuFe-MIL-101 electrochemical signals were measured and analyzed. Assay parameters were optimized, and sensitivity, stability, and linear range were assessed. Across a concentration spectrum of MUC1 spanning from 10 fg/mL to 100 ng/mL, the MB and MgAl-LDH@Fc-AuFe-MIL-101 signals were calibrated with each other, demonstrating a “signal-on-off” dual electrochemical signaling pattern. This allows for the precise and quantitative detection of MUC1 in clinical samples, offering significant potential for medical diagnosis.

TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA’s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/μL, and both assays were completed in 30min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Near-infrared light-induced homogeneous photoelectrochemical biosensor based on 3D walking nanomotor-assisted CRISPR/Cas12a for ultrasensitive microRNA-155 detection

The dysregulation of microRNA (miRNA) expression levels is intricately linked to a myriad of human diseases, and the precise and delicate detection thereof holds paramount significance in the realm of clinical diagnosis and therapy. Herein, a near-infrared (NIR) light-mediated homogeneous photoelectrochemical (PEC) biosensor was constructed for miRNA-155 detection based on NaYF4: Yb, Tm@ZnIn2S4 (NYF@ ZIS) coupled with a three-dimensional (3D) walking nanomotor-assisted CRISPR/Cas12a strategy. The upconverted light emitted by the NYF in the visible and UV region upon NIR light excitation could be utilized to excite ZIS to produce a photocurrent response. The presence of target miRNA-155 initiated an amplification reaction within the 3D walking nanomotor, resulting in the production of multiple nucleic acid fragments. These fragments could activate the collateral cleavage capability of CRISPR/Cas12a, leading to the indiscriminate cleavage of single-stranded DNA (ssDNA) on ALP-ssDNA-modified magnetic beads and the subsequent liberation of alkaline phosphatase (ALP). The released ALP facilitated the catalysis of ascorbic acid 2-phosphate to generate ascorbic acid as the electron donor to capture the photogenerated holes on the NYF@ZIS surface, resulting in a positively correlated alteration in the photocurrent response. Under optimal conditions, the NIR light-initiated homogeneous PEC biosensor had the merits of good linear range (0.1 fM to 100 pM), an acceptable limit of detection (65.77 aM) for miRNA-155 detection. Considering the pronounced sensitivity, light stability, and low photodamage, this strategy presents a promising platform for detecting various other miRNA biomarkers in molecular diagnostic practice.

Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing.

DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12-crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12-crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.

Entropy-driven reactions for controlling CRISPR/Cas12a and constructing an electrochemical biosensor for cardiac biomarkers detection.

An electrochemical biosensor is reported for controlling CRISPR/Cas12a activity through the utilization of entropy-driven reactions, alongside the construction of a highly sensitive biosensor for B-type natriuretic peptide (BNP) detection. In the biosensor, entropy-driven reactions are employed to regulate the activity of CRISPR/Cas12a-a gene editing tool-capable of nonspecific cleavage of single-stranded DNA (ssDNA). The biosensor architecture encompasses an electrode that is modified with ssDNA probes designed to hybridize with target BNP aptamers. These aptamers, furnished with labeled ssDNA triggers, facilitate the activation of CRISPR/Cas12a through interaction with its guide RNA. Upon the presence of BNP, it associates with the aptamers, subsequently liberating the triggers that instigate the entropy-driven reactions. As a consequence of these reactions, more stable duplexes emerge between the triggers and guide RNA, thereby activating CRISPR/Cas12a. The activated CRISPR/Cas12a subsequently executes cleavage of ssDNA probes residing on the electrode surface, culminating in the generation of an electrochemical signal directly (the calibration plots of differential pulse voltammetric detectionwere acquired at a working potential of 0.2V (vs. ref. electrode)) proportional to the BNP concentration. Validation of the biosensor's performance is undertaken, wherein BNP detection is demonstrated in both buffer and human serum samples. Evident in the findings is the biosensor's discernible sensitivity and specificity for BNP detection, exemplified by a detection limit of 13.53 fM and a lack of interference originating from other cardiac biomarkers, respectively. Furthermore, the biosensor's potential to discriminate between healthy individuals and those afflicted by heart failure, predicated on distinctive BNP levels, is illustrated.

Pb(II) inhibits CRISPR/Cas12a activation and application for paper-based microfluidic biosensor assisted by smartphone

A simple and low-cost paper-based microfluidic biosensor has been developed for on-site detection of Pb(II) by using CRISPR-Cas12a assisted smartphone device. Pb(II) induced G-quadruplex can inhibit the activation of the CRISPR-Cas12a system and subsequently prevent the cleavage of single-stranded DNA around gold nanoparticles. Thus, the resultant dispersed gold nanoparticles will flow into the detection zone and be subsequently captured by DNA nano-flowers in the detection zone of the paper-based microfluidic chip, resulting in the corresponding color change. The R/G values analyzed through our established SmartIons APP, correlate well with Pb(II) concentrations with a linear detection range of 0.1 μM - 10 μM and a lowest detection limit of 18.3 nM. The paper-based microfluidic biosensor exhibits high specificity, good anti-interference ability, stability and applicability. In view of low cost, easy customization and multiple channel, paper-based microfluidic biosensor has great potential for the on-site detection of Pb(II).

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo.

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

An Accurate, Rapid and Cost-Effective Method for T-nos Detection Based on CRISPR/Cas12a.

CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase terminator (T-nos) of genetically modified (GM) crops, circumventing the need for expensive instruments and technicians. For enhanced sensitivity and stability of PCR-CRISPR/Cas12a detection, we separately optimised the reaction systems for PCR amplification and CRISPR/Cas12a detection. Eleven samples of soybean samples were assessed to determine the applicability of the PCR-CRISPR/Cas12a method. The method could specifically detect target gene levels as low as 60 copies in the reaction within 50 min. In addition, accurate detection of all 11 samples confirmed the applicability. The method is not limited by large-scale instruments, making it suitable for mass detection of transgenic components in plants in the field. In conclusion, we developed a new, accurate, rapid, and cost-effective method for GM detection.

Antimicrobial activity and cytotoxicity study of cerium oxide nanoparticles with two different sizes.

The control over bacterial diseases requires the development of novel antibacterial agents. The use of antibacterial nanomedicines is one of the strategies to tackle antibiotic resistance. The study was designed to assess the antimicrobial activity of cerium oxide (CeO2 ) nanoparticles (NP) of two different sizes (CeO2 NP1 [1-2 nm] and CeO2 NP2 [10-12 nm]) and their cytotoxicity towards eukaryotic cells. The antimicrobial activity, effects of nanoparticles on DNA cleavage, microbial cell viability, and biofilm formation inhibition were analyzed. The impact of cerium oxide nanoparticles on eryptosis of erythrocytes was estimated using annexin V staining by flow cytometry. The newly synthesized CeO2 NP1 and CeO2 NP2 displayed moderate antimicrobial activities. CeO2 NP1 and CeO2 NP2 exhibited single-strand DNA cleavage ability. CeO2 NPs were found to show 100% microbial cell viability inhibition at a concentration of 500 mg/L. In addition, CeO2 NP1 and CeO2 NP2 inhibited the biofilm formation of S. aureus and P. aeruginosa. Larger cerium oxide nanoparticles were found to be less toxic against erythrocytes compared with the smaller ones. CeO2 nanoparticles demonstrate moderate antimicrobial activity and low cytotoxicity towards erythrocytes, which make them promising antibacterial agents.

A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here,we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific single-stranded DNA (ssDNA) cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target double-stranded DNA (dsDNA) substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity.

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting dsDNA phage, ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities do not significantly impact infection outcomes.

Proximity sequence enhanced CRISPR-Cas12a connected through hybridization chain reaction for sensitive biosensing of dengue virus

Dengue caused by dengue virus (DENV) is a highly pathogenic viral disease. Early detection of pathogens is very important for patient treatment and pathogen control. This work designed a dual signal amplification strategy for sensitive fluorescent detection of DENV RNA. The dual signal amplification was achieved by using a target-triggered hybridization chain reaction (HCR) to connect multiplex proximity ligation activated CRISPR-Cas12a (pCRISPR-Cas12a). The HCR was triggered by the recognition of DENV RNA to a hairpin probe (H3), which led to a product to initiate the continuous hybridization of a couple of hairpins. Here one of the hairpins (H2) was designed to contain two domains (a and b), and domains a and b from two H2 formed a proximity ligation sequence (PLS) to connect the multiplex pCRISPR-Cas12a onto the HCR product, in which domain a activated the Cas12a, and domain b enhanced the enzymatic activity by about 5 times. Upon the enzymatic cleavage of single-stranded DNA labeled with a fluorophore and quencher at each end (ssDNA-FQ), a strong fluorescent signal was produced for DENV RNA analysis, which showed a linear range from 1 pM to 10 nM and a detection limit of 51 fM. The excellent performance of the proposed homogenous fluorescent assay with high sensitivity and superior accuracy endowed this strategy broad application prospects in clinical disease diagnosis.

Characterization of Cme and Yme thermostable Cas12a orthologs

CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.

Electrochemical detection of ctDNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system

Lung cancer is the leading cause of cancer-related death around the world. The circulating tumor DNA (ctDNA) of EGFR L858R in plasma is crucial for development, targeted drug therapy, and prognosis of non-small cell lung cancer, the main type of lung cancer. Accurately detecting ctDNA using conventional methods is challenging due to its characteristics, such as considerably short size, extremely low level, and short half-life. Thus, developing a rapid, accurate, and cost-effective method for ctDNA EGFR L858R detection is urgently needed. Herein, we developed an electrochemical biosensor of ctDNA EGFR L858R based on the CRISPR/Cas12a system and MB/Fe3O4@COF/PdAu nanocomposites. The CRISPR/Cas12a system played roles in the precise recognition of ctDNA targets and indistinguishable cleavage of single-stranded DNA. Additionally, the MB/Fe3O4@COF/PdAu nanocomposite has good catalytic activity and signal amplification performance. The proposed electrochemical biosensor showed high specificity, stability, and selectivity. Notably, the limit of detection of the proposed biosensor was 3.3 aM. The detection results of 25 clinical samples showed that 22 and 20 positive samples were detected by electrochemical detection and droplet digital polymerase chain reaction, respectively. Therefore, we established a high-precision, reliable, and convenient method for ctDNA detection, which has a potential application in the diagnosis and prognosis of cancer.

CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus

SummaryCRISPR-based diagnostics (CRISPR-dx), including the Cas12-based DETECTR and Cas13-based SHERLOCK Class 2 CRISPRs, have been used to detect the presence of DNA or RNA from pathogens, such as the 2009 pandemic influenza virus A (IAV) and the 2019 novel coronavirus SARS-CoV-2. Here, we describe the collateral single-stranded DNA cleavage with Class 1 type I CRISPR-Cas3 and highlight its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection method for SARS-CoV-2. This assay, which we call Cas3-Operated Nucleic Acid detectioN (CONAN), not only detects SARS-CoV-2 in clinical samples, but also offers specific detection of single-base-pair mutations in IAV variants. This tool allows rapid and accurate point-of-care testing for patients with suspected SARS-CoV-2 or drug-resistant IAV infections in hospitals.

The study on biological activities of silver nanoparticles produced via green synthesis method using Salvia officinalis and Thymus vulgaris.

In the present study, Ag nanoparticles (AgNPs) were synthesized from Salvia officinalis and Thymus vulgaris, known as phytotherapy plants. The obtained silver nanoparticles were characterized using SEM, XRD, FTIR, and UV/Vis spectra. The antioxidant capacities of Salvia officinalis-mediated AgNP (SO-AgNP) and Thymus vulgaris-mediated AgNP (TV-AgNP) were analyzed in vitro using 1,1-diphenyl-2-picrylhydrazyl and iron chelating activity assays. DPPH activities were 83.74% and 57.17% for SO-AgNP and TV-AgNP at concentration 200 mg/L, respectively. Both green synthesized AgNPs exhibited good iron chelating activity. In addition, the DNA cleavage activities of SO-AgNPs and TV-AgNP were investigated with agarose gel electrophoresis technique. SO-AgNPs and TV-AgNP showed single-strand DNA cleavage activity. AgNPs showed that the SO-AgNP and TV-AgNp were effective against bacteria and fungi, and antimicrobic activities were assessed as minimal inhibition concentration (MIC). Remarkably, green synthesized AgNPs showed highly effective cell viability and biofilm inhibition effect. AgNPs also demonstrated slightly antimicrobial photodynamic activity after LED irradiation.

CRISPR-/Cas12a-Mediated Liposome-Amplified Strategy for the Surface-Enhanced Raman Scattering and Naked-Eye Detection of Nucleic Acid and Application to Food Authenticity Screening

Surface-enhanced Raman scattering (SERS) has been recognized as a powerful tool for biosensors due to the ultrahigh sensitivity and unique fingerprint information. However, there are some limitations in trace target nucleic acid detection for the restricted signal-transducing and amplification strategies. Inspired by CRISPR/Cas12a with specific target DNA-activated collateral single-strand DNA (ssDNA) cleavage activity and liposome with signal molecule-loading properties, we first proposed a sensitive SERS-based on-site nucleic acid detection strategy mediated by CRISPR/Cas12a with trans-cleavage activity on ssDNA linkers utilized to capture liposomes. Liposomes loading two kinds of signal molecules, 4-nitrothiophenol (4-NTP) and cysteine, could achieve the dual-mode detection of target DNA with SERS and naked eye, respectively. The promptly amplified signals were initiated by the triggered breakdown of signal molecule-loaded liposomes. Emancipated 4-NTP, a biological-silent Raman reporter, would achieve highly selective and sensitive SERS measurement. Released cysteine induced the aggregation of plasmonic gold nanoparticles, leading to an obvious red to blue colorimetric shift to realize portable naked-eye detection. With this strategy, target nucleic acid concentration was dexterously converted into SERS and visualization signals and could be detected as low as 100 aM and 10 pM, respectively. The approach was also successfully applied to determine meat adulteration, achieving the detection of a low adulteration ratio in the complicated food matrix. We anticipate that this strategy will not only be regarded as a universal platform for the on-site detection of food authenticity but also broaden SERS application for the accurate determination of diverse biomarkers.

Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection

Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.