Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus.

Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay's performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.

Hydrophilic/hydrophobic modified microchip for detecting multiple gene doping candidates using CRISPR-Cas12a and RPA.

With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.

Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA.

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.

Analysis of Whole-Genome for Identification of Seven Penicillium Species with Significant Economic Value.

The Penicillium genus exhibits a broad global distribution and holds substantial economic value in sectors including agriculture, industry, and medicine. Particularly in agriculture, Penicillium species significantly impact plants, causing diseases and contamination that adversely affect crop yields and quality. Timely detection of Penicillium species is crucial for controlling disease and preventing mycotoxins from entering the food chain. To tackle this issue, we implement a novel species identification approach called Analysis of whole GEnome (AGE). Here, we initially applied bioinformatics analysis to construct specific target sequence libraries from the whole genomes of seven Penicillium species with significant economic impact: P. canescens, P. citrinum, P. oxalicum, P. polonicum, P. paneum, P. rubens, and P. roqueforti. We successfully identified seven Penicillium species using the target we screened combined with Sanger sequencing and CRISPR-Cas12a technologies. Notably, based on CRISPR-Cas12a technology, AGE can achieve rapid and accurate identification of genomic DNA samples at a concentration as low as 0.01 ng/µL within 30 min. This method features high sensitivity and portability, making it suitable for on-site detection. This robust molecular approach provides precise fungal species identification with broad implications for agricultural control, industrial production, clinical diagnostics, and food safety.

Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

Probiotic bacterial adsorption coupled with CRISPR/Cas12a system for mercury (II) ions detection.

The complex sample matrix poses significant challenges in accurately detecting heavy metals. In view of its superior performance for the biological adsorption of heavy metals, probiotic bacteria can be explored for functional unit to eliminate matrix interference. Herein, Lactobacillus rhamnosus (LGG) demonstrates a remarkable tolerance and can adsorb up to 300 µM of Hg2+, following the Freundlich isotherm model with the correlation coefficient (R2) value of 0.9881. Subsequently, by integrating the CRISPR/Cas12a system, a sensitive and specific fluorescent biosensor, "Cas12a-MB," has been developed for Hg2+ detection. Specifically, Hg2+ adsorbed onto LGG can specifically bind to the nucleic acid probe, thereby inhibiting the binding of the probe to LGG and the subsequent activation of the CRISPR/Cas12a system. Under optimal experimental conditions, with the detection time of 90 min and the detection limit of 0.44 nM, the "Cas12a-MB" biosensor offers a novel, eco-friendly approach for Hg2+ detection, showcasing the innovative application of probiotics in biosensor.

Visual detection of fungicide resistance by combining RPA and CRISPR/Cas12a in peach Brown rot fungus Monilinia fructicola.

BACKGROUND: Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice. RESULTS: In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the ß-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains. CONCLUSION: Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry.

A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations.

Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs). In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing. In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.

Electrochemical sensing technology based on a ligation-initiated LAMP-assisted CRISPR/Cas12a system for high-specificity detection of EGFR E746-A750 deletion mutation.

Epidermal growth factor receptor (EGFR) mutation status is pivotal in predicting the efficacy of tyrosine kinase inhibitor treatments against tumors. Among EGFR mutations, the E746-A750 deletion is particularly common and accurately quantifying it can guide targeted therapies. This study introduces a novel visual sensing technology using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system guided by ligation-initiated loop-mediated isothermal amplification (LAMP) to detect the del E746-A750 mutation in EGFR. Conventional LAMP primers were simplified by designing a pair of target-specific stem-loop DNA probes, enabling selective amplification of the target DNA. The CRISPR/Cas12a system was employed to identify the target nucleic acid and activate Cas12a trans-cleavage activity, thereby enhancing the specificity of the assay. Furthermore, the biosensor utilized high-performance nanomaterials such as triangular gold nanoparticles and graphdiyne, known for their large specific surface area, to enhance sensitivity effectively as a sensing platform. The proposed biosensor demonstrated outstanding specificity, achieving a low detection limit of 17 fM (S/N = 3). Consequently, this innovative strategy not only expands the application scope of CRISPR/Cas12a technology but also introduces a promising approach for clinical diagnostics in modern medicine.

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.

A novel system with robust compatibility and stability for detecting Sugarcane yellow leaf virus based on CRISPR-Cas12a.

Sugarcane yellow leaf virus (SCYLV) can reduce sugarcane productivity. A novel detection system based on reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) combined with CRISPR-Cas12a, named RT-MIRA-CRISPR-Cas12a, was developed. This innovative approach employs crude leaf extract directly as the reaction template, streamlining the extraction process for simplicity and speed. Combining RT-MIRA and CRISPR-Cas12a in one reaction tube increases the ease of operation while reducing the risk of aerosol contamination. In addition, it exhibits sensitivity equivalent to qPCR, boasting a lower detection limit of 25 copies. Remarkably, the entire process, from sample extraction to reaction completion, requires only 52-57 minutes, just a thermostat water bath. The result can be observed and judged by the naked eye.IMPORTANCESugarcane yellow leaf disease (SCYLD) is an important viral disease that affects sugarcane yield. There is an urgent need for rapid, sensitive, and stable detection methods. The reverse transcription-multienzyme isothermal rapid amplification combined with CRISPR-Cas12a (RT-MIRA-CRISPR-Cas12a) method established in this study has good specificity and high sensitivity. In addition, the system showed good compatibility and stability with the crude leaf extract, as shown by the fact that the crude extract of the positive sample could still be stably detected after 1 week when placed at 4°C. RT-MIRA-CRISPR-Cas12a, reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect SCYLV on 33 sugarcane leaf samples collected from the field, and it was found that the three methods reached consistent conclusions. This Cas12a-based detection method proves highly suitable for the rapid on-site detection of the SCYLV.

RPA-CRISPR/Cas12a-mediated isothermal amplification for rapid detection of Phytopythium helicoides.

Phytopythium helicoides, which belongs to the algae (Chromista), Oomycota, Pythiales, Pythiaceae and Phytophthora, is a quarantine pathogen that causes brown rot of fruits, stem rot and root rot, along with other symptoms that can damage several tree species in urban landscaping. Therefore, disease management requires rapid and accurate diagnosis. The present study used recombinase polymerase amplification (RPA) in conjunction with the CRISPR/Cas12a system to identify P. helicoides. The test exhibited high specificity and sensitivity and could detect 10 pg.µL-1 of P. helicoides genomic DNA at 37 ℃ within 20 minutes. The test results were visible by excitation of fluorophores by blue light. This groundbreaking test is able to detect P. helicoides in artificially inoculated Rhododendron leaves. The RPA-CRISPR/Cas12a detection assay developed in this study is characterized by its sensitivity, efficiency, and convenience. Early detection and control of P. helicoides is crucial for the protection of urban green cover species.

Application of CRISPR/Cas12a in miRNA-155 detection: A novel homogeneous electrochemiluminescence biosensor.

BACKGROUND: MicroRNAs (miRNAs) are important non-coding RNA entities that affect gene expression and function by binding to target mRNAs, leading to degradation of the mRNAs or inhibiting their translation. MiRNAs are widely involved in a variety of biological processes, such as cell differentiation, development, metabolism, and apoptosis. In addition, miRNAs are associated with many diseases, including cancer. However, conventional detection techniques often suffer from shortcomings such as low sensitivity, so we need to develop a rapid and efficient detection strategy for accurate detection of miRNAs. RESULTS: We have developed an innovative homogeneous electrochemiluminescence (ECL) biosensor. This biosensor employs CRISPR/Cas12a gene editing technology for accurate and efficient detection of microRNA (miRNA). Compared to conventional technologies, this biosensor employs a unique homogeneous detection format that eliminates laborious probe fixation steps and greatly simplifies the detection process. By using two amplification techniques - isothermal amplification and T7 RNA polymerase amplification - the biosensor improves the sensitivity and specificity of the assay, providing excellent detection performance in the assay. This makes it possible to evaluate miRNA directly from a variety of biological samples such as cell lysates and diluted human serum. Experimental results convincingly demonstrate the extraordinary performance of this biosensor, including its extremely low detection limit of 1.27 aM, high sensitivity, reproducibility and stability. SIGNIFICANCE: The application of our constructed sensor in distinguishing between cancerous and non-cancerous cell lines highlights its potential for early cancer detection and monitoring. This innovative approach represents a major advancement in the field of miRNA detection, providing a user-friendly, cost-effective, and sensitive solution with broad implications for clinical diagnosis and patient care, especially in point-of-care settings.

Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR/Cas12a.

Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/µL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.

Specific detection of gut pathogens for one-pot chip based on RPA-CRISPR/Cas12a.

BACKGROUND: There are billions of bacteria in the intestine, most of which are harmless and play important roles in humans. Although only a very small number of bacteria can cause diseases, once the pathogenic bacteria are ingested into the body and multiply in large quantities, it can lead to inflammatory diseases in the intestines and even other organs. Although polymerase chain reaction can specifically detect bacterial nucleic acid. However, the demand for temperature cycling limits its portability. Therefore, it is hoped to establish a high-throughput, highly specific and portable detection platform for directly detecting nucleic acid of intestinal pathogens. RESULTS: Herein, a one-pot chip based on RPA-CRCISPR/Cas12a platform was developed. The chip is the same size as a glass slide and allows detection at the same temperature. Multiple samples could be detected simultaneously on the one chip, achieved high-throughput detection and improved the integration of detection. The specific recognition of CRISPR/Cas12a avoided the influence of non-specific amplification of RPA and enhanced the specificity of the analysis. At the same time, the one-pot chip avoided secondary contamination when the lid was opened during the analysis process. And the bacterial concentration showed good linearity at 102-108 cfu mL-1. The limit of detection could be as low as 0.43 cfu mL-1. This method has been successfully used to detect pollution samples. It can provide a reliable platform for early screening of gastrointestinal and other inflammatory diseases. SIGNIFICANCE: The one-pot chip based on the RPA-CRISPR/Cas12a platform established can directly detect the nucleic acid of intestinal pathogens, with portability and specificity. It is worth noting that the platform has good programmability, can be used for other target detection by changing crRNA and RPA primers, it can achieve multi sample detection on the one chip.

The clinical value of rapidly detecting urinary exosomal lncRNA RMRP in bladder cancer with an RT-RAA-CRISPR/Cas12a method.

BACKGROUND AND AIMS: Bladder cancer (BCa) is a highly aggressive malignancy of the urinary system. Timely detection is imperative for enhancing BCa patient prognosis. MATERIALS AND METHODS: This study introduces a novel approach for detecting long non-coding RNA (lncRNA) Mitochondrial RNA Processing Endoribonuclease (RMRP) in urine exosomes from BCa patients using the reverse transcription recombinase-aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats and associated Cas12a proteins (CRISPR/Cas12a) technique. Various statistical methods were used to evaluate its diagnostic value for BCa. RESULTS: The specificity of urine exosomal RMRP detection for BCa diagnosis was enhanced by using RT-RAA combined with CRISPR/Cas12a. The testing process duration was reduced to 30 min, which supports rapid detection. Moreover, this approach allows the identification of target signals in real-time using blue light, facilitating immediate detection. In clinical sample analysis, this methodology exhibited a high level of diagnostic efficacy. This was evidenced by larger area under the curve values with receiver operating characteristic curve analysis compared with using traditional RT-qPCR methods, indicating superior diagnostic accuracy and sensitivity. Furthermore, the combined analysis of RMRP expression in urine exosomes detected by RT-RAA-CRISPR/Cas12a and NMP-22 expression may further enhance diagnostic accuracy. CONCLUSIONS: The RT-RAA-CRISPR/Cas12a technology is a swift, sensitive, and uncomplicated method for nucleic acid detection. Because of its convenient and non-invasive sampling approach, user-friendly operation, and reproducibility, this technology is very promising for automated detection and holds favorable application possibilities within clinical environments.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

A rapid, ultrasensitive, and highly specific method for detecting fowl adenovirus serotype 4 based on the LAMP-CRISPR/Cas12a system.

Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium hepatitis syndrome in chickens, which causes severe economic impact to the poultry industry. A simple, swift and reliable detection is crucial for timely identification of FAdV-4 infection, promoting effective viral prevention and control measures. Herein, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system detection platform based on loop-mediated isothermal amplification (LAMP) was studied. The CRISPR RNA (crRNA) and LAMP primers were designed and screened based on the highly conserved region of the FAdV-4 hexon gene. The parameters were then optimized individually to achieve the ideal reaction performance. The platform could lead visual detection of FAdV-4 to achieve as low as 1 copy in less than 40 min without the need for specialized instrumentation or complex equipment. Moreover, it was greatly specific, and did not cross-react with other common avian viruses. Following the validation of 30 clinical samples of suspected FAdV-4 infection, the results LAMP-CRISPR/Cas12a method generated showed fully concordance with which of the gold standard quantitative real-time PCR. To summarize, this study presented a novel, swift, expedient and inexpensive detection platform for FAdV-4, which is beneficial to viral inchoate diagnosis and point-of-care testing.

One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.

Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 µL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.