Velcrin compounds activate the SLFN12 tRNase to induce tomoptosis.

Velcrins are molecular glues that induce complex formation between PDE3A and SLFN12. The PDE3A-SLFN12 complex activates the SLFN12 RNase, resulting in cleavage of the specific substrate, tRNA-Leu-TAA, global inhibition of translation, and death of cells expressing sufficient levels of both proteins. Here, unanswered questions about the mechanism of action and therapeutic promise of velcrin compounds are discussed.

Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum.

The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations such as large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In this study, we aim to elucidate the unique protein features associated with Cas9 and Cas12 families and identify the features distinguishing each family from non-Cas proteins. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,494 features) encoding various physiochemical, topological, constitutional, and coevolutionary information on Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and non-Cas proteins. All the models were evaluated rigorously on the test and independent data sets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 92% and 95% on their respective independent data sets, while the multiclass classifier achieved an F1 score of close to 0.98. We observed that Quasi-Sequence-Order (QSO) descriptors like Schneider.lag and Composition descriptors like charge, volume, and polarizability are predominant in the Cas12 family. Conversely Amino Acid Composition descriptors, especially Tripeptide Composition (TPC), predominate the Cas9 family. Four of the top 10 descriptors identified in Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all Cas9 proteins and located within different catalytically important domains of the Streptococcus pyogenes Cas9 (SpCas9) structure. Among these, DHI and HHA are well-known to be involved in the DNA cleavage activity of the SpCas9 protein. Mutation studies have highlighted the significance of the PWN tripeptide in PAM recognition and DNA cleavage activity of SpCas9, while Y450 from the PYY tripeptide plays a crucial role in reducing off-target effects and improving the specificity in SpCas9. Leveraging our machine learning (ML) pipeline, we identified numerous Cas9 and Cas12 family-specific features. These features offer valuable insights for future experimental and computational studies aiming at designing Cas systems with enhanced gene-editing properties. These features suggest plausible structural modifications that can effectively guide the development of Cas proteins with improved editing capabilities.

CRISPR/Cas12a trans-cleavage mediated photoelectrochemical biosensor based on zeolitic imidazolate framework-67 for ATP determination.

An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating CRISPR/Cas12a trans-cleavage activity and specific recognition of ZIF-67 to ATP. Exciton energy transfer between CdTe QDs and AuNPs system is firstly constructed as photoelectrochemical (PEC) sensing substrate. Then, the activator DNAs, used to activate CRISPR/Cas12a, are absorbed on the surface of ZIF-67. In the presence of ATP, the activator DNAs are released due to more efficient adsorption of ZIF-67 to ATP. The released activator DNA activates trans-cleavage activity of CRISPR/Cas12a to degrade ssDNA on the electrode, leading to the recovery of photocurrent due to the interrupted energy transfer. Benefiting from the specific recognition of ZIF-67 to ATP and CRISPR/Cas12a-modulated amplification strategy, the sensor is endowed with excellent specificity and high sensitivity.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A.

BACKGROUND: Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread. METHODS: In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system. RESULTS: The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours. CONCLUSIONS: Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

A highly specific and ultrasensitive approach to detect Prymnesium parvum based on RPA-CRISPR-LbaCas12a-LFD system.

BACKGROUND: Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance. RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL). SIGNIFICANCE: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.

Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA.

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 µL in gargle and 166 viral particles/100 µL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

A universal electrochemical biosensor based on CRISPR/Cas12a and a DNA tetrahedron for ultrasensitive nucleic acid detection.

Herein, a universal nucleic acid analysis platform was constructed for sensitive and accurate detection of miRNA-155 and ctDNA using isothermal amplification-assisted CRISPR/Cas12a and a tetrahedral DNA nanostructure (TDN) supported sensing interface. Under the optimal experimental conditions, the prepared sensor achieved specific detection of miRNA-155 and ctDNA at as low as aM levels in 2.6 h. Furthermore, the platform was also successfully applied to human serum sample recovery experiments and cancer cell lysates, demonstrating outstanding reliability and accuracy. We firmly believe that this work provides a universal, sensitive, and practical tool for early clinical diagnosis.

CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing.

CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.

A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

CRISPR/Cas12a-mediated DNA-AgNC label-free logical gate for multiple microRNAs' assay.

CRISPR/Cas system has been widely applied in the assay of disease-related nucleic acids. However, it is still challenging to use CRISPR/Cas system to detect multiple nucleic acids at the same time. Herein, we combined the preponderance of DNA logic circuit, label-free, and CRISPR/Cas technology to construct a label-free "AND" logical gate for multiple microRNAs detection with high specificity and sensitivity. With the simultaneous input of miRNA-155 and miRNA-141, the logic gate starts, and the activation chain of Cas12a is destroyed; thus, the activity is inhibited and the fluorescence of the signal probe ssDNA-AgNCs is turned on. The detection limit of this method for simultaneous quantitative detection of double target is 84 fmol/L (S/N = 3). In this "AND" logic gate, it is only necessary for the design of a simple DNA hairpin probe, which is inexpensive and easy, and since this method involves only one signal output, the data processing is very simple. What is more important, in this strategy two types of microRNAs can be monitored simultaneously by only using CRISPR/Cas12a and a type of crRNA, which offers a new design concept for the exploitation of single CRISPR/Cas system for multiple nucleic acid assays.

Cascade CRISPR/Cas12a and DSN for the electrochemical biosensing of miR-1246 in BC-derived exosomes.

MiR-1246 in breast cancer-derived exosomes was a promising biomarker for early diagnosis of breast cancer(BC). However, the low abundance, high homology and complex background interference make the accurate quantitative detection of miR-1246 facing great challenges. In this study, we developed an electrochemical biosensor based on the subtly combined of CRISPR/Cas12a, double-stranded specific nuclease(DSN) and magnetic nanoparticles(MNPs) for the detection of miR-1246 in BC-derived exosomes. Ascribed to the good synergistic effect of DSN, Cas12a and MNPs, the developed electrochemical biosensor exhibited excellent performance with the linear range from 500 aM to 5 pM, and the detection limit as low down to about 50 aM. The target-specific triggered enzyme-digest activity of DSN and Cas12a system, as well as the powerful separation ability of MNPs ensure the high specificity of developed electrochemical biosensor which can distinguish single base mismatches. In addition, the developed electrochemical biosensor has been successfully applied to detect miR-1246 in blood-derived exosomes and realize distinguishing the BC patients from the healthy individuals. It is expected that the well-designed biosensing platform will open up new avenues for clinical liquid biopsy and early screening of breast cancer, as well as provide deeper insights into clinical oncology treatment.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Sensitive aptasensing of ATP based on a PAM site-regulated CRISPR/Cas12a activation.

The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.

Harnessing noncanonical crRNA for highly efficient genome editing.

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system.

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA.

BACKGROUND: CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. RESULTS: Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. SIGNIFICANCE: The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

A colorimetric tandem combination of CRISPR/Cas12a with dual functional hybridization chain reaction for ultra-sensitive detection of Mycobacterium bovis.

Tuberculosis caused by Mycobacterium bovis poses a global infectious threat to humans and animals. Therefore, there is an urgent need to develop a sensitive, precise, and easy-to-readout strategy. Here, a novel tandem combination of a CRISPR/Cas12a system with dual HCR (denoted as CRISPR/Cas12a-D-HCR) was constructed for detecting Mycobacterium bovis. Based on the efficient trans-cleavage activity of the active CRISPR/Cas12a system, tandem-dsDNA with PAM sites was established using two flexible hairpins, providing multiple binding sites with CRISPR/Cas12a for further amplification. Furthermore, the activation of Cas12a initiated the second hybridization chain reaction (HCR), which integrated complete G-quadruplex sequences to assemble the hemin/G-quadruplex DNAzyme. With the addition of H2O2 and ABTS, a colorimetric signal readout strategy was achieved. Consequently, CRISPR/Cas12a-D-HCR achieved a satisfactory detection linear range from 20 aM to 50 fM, and the limit of detection was as low as 2.75 aM with single mismatched recognition capability, demonstrating good discrimination of different bacterial species. Notably, the practical application performance was verified via the standard addition method, with the recovery ranging from 96.0% to 105.2% and the relative standard deviations (RSD) ranging from 0.95% to 6.45%. The proposed CRISPR/Cas12a-D-HCR sensing system served as a promising application for accurate detection in food safety and agricultural fields.