A CRISPR-Cas12a-based electrochemical biosensor for the detection of microphthalmia-associated transcription factor.

A novel electrochemical biosensor that combines the CRISPR-Cas12a system with a gold electrode is reported for the rapid and sensitive detection of microphthalmia-associated transcription factor (MITF). The biosensor consists of a gold electrode modified with DNA1, which contains the target sequence of MITF and is labeled with ferrocene, an electroactive molecule. The biosensor also includes hairpin DNA, which has a binding site for MITF and can hybridize with helper DNA to form a double-stranded complex that activates CRISPR-Cas12a. When MITF is present, it binds to hairpin DNA and prevents its hybridization with helper DNA, thus inhibiting CRISPR-Cas12a activity and preserving the DPV signal of ferrocene. When MITF is absent, hairpin DNA hybridizes with helper DNA and activates CRISPR-Cas12a, which cleaves DNA1 and releases ferrocene, thus reducing the DPV signal. The biosensor can detect MITF with high sensitivity (with an LOD of 8.14 fM), specificity, and accuracy in various samples, such as cell nuclear extracts and human serum. The biosensor can also diagnose and monitor melanocyte-related diseases and melanin production. This work provides a simple, fast, sensitive, and cost-effective biosensor for MITF detection and a valuable tool for applications in genetic testing, disease diagnosis, and drug screening.

A novel electrochemical aptasensor based on NrGO-H-Mn3O4 NPs integrated CRISPR/Cas12a system for ultrasensitive low-density lipoprotein determination.

Atherosclerosis cardiovascular disease (ASCVD) has become one of the leading death causes in humans. Low-density lipoprotein (LDL) is an important biomarker for assessing ASCVD risk level. Thus, monitoring LDL levels can be an important means for early diagnosis of ASCVD. Herein, a novel electrochemical aptasensor for determination LDL was designed based on nitrogen-doped reduced graphene oxide-hemin-manganese oxide nanoparticles (NrGO-H-Mn3O4 NPs) integrated with clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR/Cas12a) system. NrGO-H-Mn3O4 NPs not only have a large surface area and remarkable enhanced electrical conductivity but also the interconversion of different valence states of iron in hemin can provide an electrical signal. Nonspecific single-stranded DNA (ssDNA) was bound to NrGO-H-Mn3O4 NPs to form a signaling probe and was immobilized on the electrode surface. The CRISPR/Cas12a system has excellent trans-cleavage activity, which can be used to cleave ssDNA, thus detaching the NrGO-H-Mn3O4 NPs from the sensing interface and attenuating the electrical signal. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the LDL in range from 0.005 to 1000.0 nM with the detection limit of 0.005 nM. The proposed sensor exhibited good stability, selectivity, and stability and achieved reliable detection of LDL in serum samples, demonstrating its promising application prospects for the diagnostic application of LDL.

Development of a separation platform comprising magnetic beads combined with the CRISPR/Cas12a system enabling ultrasensitive and rapid detection of miRNA-21.

A separation platform has been developed mediated by a combination of magnetic beads and the CRISPR/Cas12a system to achieve ultrasensitive and rapid detection of miRNA-21 at a low level. In this system, with the assistance of an auxiliary probe, the target miRNA-21 can be specifically combined with three-stranded probes to initiate the SDR reaction. Abundant aptamer A3 was added to the solution that can activate the CRISPR/Cas12a system and initiate the trans-cleavage reaction to recover the fluorescence signal. Using magnetic beads to mediate the separation considerably greatly improves the signal conversion efficiency and detection sensitivity. At the 492 nm excitation wavelength, and 502-650 nm scan range, through analyzing the fluorescence peak intensity at 520 nm, the biosensor's determination range and limit of detection is 8 fM-250 nM and 2.42 fM, respectively, and the RSD is 19.03-37.80. Compared with other biosensors, the biosensor developed exhibited superior performance and the signal recovered excellently in 1% human serum and the LOD is 12.12 fM. This method provides a novel highly sensitive scheme for detecting miRNA .

Modularization of dual recognized CRISPR/Cas12a system for the detection of Staphylococcus aureus assisted by hydrazone chemistry.

In this work, a dual recognized CRISPR/Cas12a system has been proposed, in which the activation chain is cleverly divided into two parts that can serve for precise dual target recognition, and hydrazone chemistry is introduced for the formation of a whole activation chain. It has been further explored to construct a new method for the specific and sensitive detection of Staphylococcus aureus (SA) as one of the most common pathogens in infectious diseases. In virtue of proximity effect contributed by complementary base pairing, hydrazone chemistry accelerates the formation of the whole activation strand and improves the specificity of the CRISPR/Cas12a system, serving for the accurate analysis of SA. Moreover, the temporary aggregation of CRISPR/Cas12a around SA enhances its catalytical efficiency so as to further amplify signal. With high sensitivity, stability, reproducibility and specificity, the established method has been successfully applied to detect SA in complex substrates. Meanwhile, our established method can well evaluate the inhibition effect of chlorogenic acid and congo red in comparison with flow cytometry. ENVIRONMENTAL IMPLICATION: Bacterial pathogens exist widely in the environment and seriously threaten the safety of human health. Staphylococcus aureus (SA) is the most common pathogen of human suppurative infection, which can cause local suppurative infection, pneumonia, and even systemic infections such as sepsis. In this work, a dual recognized CRISPR/Cas12a system mediated by hydrazone chemistry has been proposed. With high sensitivity and low detection limit, the established method can specifically detect SA and effectively evaluate the antibacterial effect of inhibitors. This method is expected to be further developed into a detection method in different scenarios such as environmental monitoring and clinical diagnosis.

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.

High-Throughput and Integrated CRISPR/Cas12a-Based Molecular Diagnosis Using a Deep Learning Enabled Microfluidic System.

CRISPR/Cas-based molecular diagnosis demonstrates potent potential for sensitive and rapid pathogen detection, notably in SARS-CoV-2 diagnosis and mutation tracking. Yet, a major hurdle hindering widespread practical use is its restricted throughput, limited integration, and complex reagent preparation. Here, a system, microfluidic multiplate-based ultrahigh throughput analysis of SARS-CoV-2 variants of concern using CRISPR/Cas12a and nonextraction RT-LAMP (mutaSCAN), is proposed for rapid detection of SARS-CoV-2 and its variants with limited resource requirements. With the aid of the self-developed reagents and deep-learning enabled prototype device, our mutaSCAN system can detect SARS-CoV-2 in mock swab samples below 30 min as low as 250 copies/mL with the throughput up to 96 per round. Clinical specimens were tested with this system, the accuracy for routine and mutation testing (22 wildtype samples, 26 mutational samples) was 98% and 100%, respectively. No false-positive results were found for negative (n = 24) samples.

Enhancing CRISPR/Cas-mediated electrochemical detection of nucleic acid using nanoparticle-labeled covalent organic frameworks reporters.

Molecular detection of nucleic acid plays an important role in early diagnosis and therapy of disease. Herein, a novel and enhanced electrochemical biosensor was exploited based on target-activated CRISPR/Cas12a system coupling with nanoparticle-labeled covalent organic frameworks (COFs) as signal reporters. Hollow spherical COFs (HCOFs) not only served as the nanocarriers of silver nanoparticles (AgNPs)-DNA conjugates for enhanced signal output but also acted as three-dimensional tracks of CRISPR/Cas12a system to improve the cleavage accessibility and efficiency. The presence of target DNA triggered the trans-cleavage activity of the CRISPR/Cas12a system, which rapidly cleaved the AgNPs-DNA conjugates on HCOFs, resulting in a remarkable decrease of the electrochemical signal. As a proof of concept, the fabricated biosensing platform realized highly sensitive and selective detection of human papillomavirus type 16 (HPV-16) DNA ranging from 100 fM to 1 nM with the detection limit of 57.2 fM. Furthermore, the proposed strategy provided a versatile and high-performance biosensor for the detection of different targets by simple modification of the crRNA protospacer, holding promising applications in disease diagnosis.

Self-assembled bifunctional nanoflower-enabled CRISPR/Cas biosensing platform for dual-readout detection of Salmonella enterica.

Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica. Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 101-108 cfu/mL and 102 cfu/mL (lateral flow assay) in the linear range of 102-108 cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica. This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens.

Detection of biological loads in sewage using the automated robot-driven photoelectrochemical biosensing platform.

Real-time polymerase chain reaction (RT-PCR) remains the most prevalent molecular detection technology for sewage analysis but is plagued with numerous disadvantages, such as time consumption, high manpower requirements, and susceptibility to false negatives. In this study, an automated robot-driven photoelectrochemical (PEC) biosensing platform is constructed, that utilizes the CRISPR/Cas12a system to achieve fast, ultrasensitive, high specificity detection of biological loads in sewage. The Shennong-1 robot integrates several functional modules, involving sewage sampling and pretreatment to streamline the sewage monitoring. A screen-printed electrode is employed with a vertical graphene-based working electrode and enhanced with surface-deposited Au nanoparticles (NPs). CdTe/ZnS quantum dots (QDs) are further fabricated through the double-stranded DNA (dsDNA) anchored on Au NPs. Using the cDNA template of Omicron BA.5 spike gene as a model, the PEC biosensor demonstrates excellent analytical performance, with a lower detection limit of 2.93 × 102 zm and an outstanding selectivity at the level of single-base mutation recognition. Furthermore, the rapid, accurate detection of BA.5 in sewage demonstrates the feasibility of the PEC platform for sewage monitoring. In conclusion, this platform allows early detection and tracking of infectious disease outbreaks, providing timely data support for public health institutions to take appropriate prevention and control measures.

Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3.

The porcine circovirus type 3 (PCV3) infection is an emerging disease associated with clinical signs of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs. Currently, there is a lack of effective vaccines and therapeutics against this disease. Therefore, rapid, effective, sensitive, and specific detection methods are crucial for the timely identification, prevention, and control of PCV3. In this study, we developed one- and two-pot visual detection methods for PCV3 using a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection system combined with recombinase polymerase amplification (RPA). These two methods demonstrated no cross-reactivity with eight other swine viruses and exhibited minimum detection limits of five and two copies of viral DNA, respectively, revealing their high specificity and sensitivity. During a clinical sample detection within 30 min, the coincidence rates between the one- and two-pot detection methods and real-time quantitative polymerase chain reaction (qPCR) were 100%. In conclusion, both one- and two-pot RPA-CRISPR/Cas12a detection methods have significant potential for the rapid, sensitive, and specific visual detection of PCV3.

High-throughput identification of meat ingredients in adulterated foods based on centrifugal integrated purification-CRISPR array.

Rapid, accurate, and sensitive analytical methods for the detection of food fraud are now an urgent requirement in the global food industry to ensure food quality. In response to this demand, a centrifugal integrated purification-CRISPR array for meat adulteration (CIPAM) was established. In detail, CIPAM system combines microneedles for DNA extraction and RAA-CRISPR/Cas12a integrated into a centrifugal microfluidic chip for the detection of meat adulteration. The RAA-CRISPR/Cas12a reaction reagents were pre-embedded into the different reaction chambers on the microfluidic chip to achieve the streamline of operations, markedly simplifying the detection process. The whole reaction was completed within 30 min with a detection limit of 0.1 % (w/w) in pig, chicken, duck, and lamb products. Referring to the results of the standard method, CIPAM system achieved 100 % accuracy. The automatic multiplex detection process implemented in the developed CIPAM system met the needs of food regulatory authorities.

Infectious Disease Diagnosis and Pathogen Identification Platform Based on Multiplex Recombinase Polymerase Amplification-Assisted CRISPR-Cas12a System.

Controlling and mitigating infectious diseases caused by multiple pathogens or pathogens with several subtypes require multiplex nucleic acid detection platforms that can detect several target genes rapidly, specifically, sensitively, and simultaneously. Here, we develop a detection platform, termed Multiplex Assay of RPA and Collateral Effect of Cas12a-based System (MARPLES), based on multiplex nucleic acid amplification and Cas12a ssDNase activation to diagnose these diseases and identify their pathogens. We use the clinical specimens of hand, foot, and mouth disease (HFMD) and influenza A to evaluate the feasibility of MARPLES in diagnosing the disease and identifying the pathogen, respectively, and find that MARPLES can accurately diagnose the HFMD associated with enterovirus 71, coxsackievirus A16 (CVA16), CVA6, or CVA10 and identify the exact types of H1N1 and H3N2 in an hour, showing high sensitivity and specificity and 100% predictive agreement with qRT-PCR. Collectively, our findings demonstrate that MARPLES is a promising multiplex nucleic acid detection platform for disease diagnosis and pathogen identification.

Customization of aptamer to develop CRISPR/Cas12a-derived ultrasensitive biosensor.

The CRISPR/Cas systems have provided wide biosensing applications. Particularly, the aptamer-involved CRISPR/Cas sensor system powerfully expanded to non-nucleic-acid targets. However, tailoring the sequence of the aptamer to explore the relationship between affinity and the activation of CRISPR/Cas12a trans-cleavage activity has not been reported yet. Herein, we developed a series of new aptamers toward the spike protein 1(S1) of SARS-CoV-2. Surface plasmon resonance measurements showed that the affinity of these aptamers to S1 was at the nM level. Subsequently, a "SET" effect (Sequence Essential Trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, an aptamer, as the activator, sequence needs to be tailored to activate CRISPR/Cas12a efficiently. A balance should be reached between affinity and activation ability. On the one hand, high affinity ensures target recognition performance, and on the other hand, activation can achieve adequate amplification and output of recognition signals. The optimized sequence (with 27 nucleotides, for short 27-nt) not only recognizes the target with a high affinity and specificity but also can trigger the CRISPR/Cas12a trans-cleavage activity efficiently, showing an excellent detection performance in electrochemical biosensors. The detection limit for SARS-CoV-2 S1 can be low at 1.5 pg mL-1. The new CRISPR/Cas12a-derived aptasensor also displays a remarkable ability to detect Beta, Delta, and Omicron variants but is selective toward other kinds of proteins. Above all, it is robust for point-of-care testing (POCT) in complex biological fluids, such as saliva, urine, and serum, and provides a universal and scalable detecting platform. Our results provide new insights into aptamer development and a different strategy for COVID-19 antigen detection and biosensor development.

Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.

RPA-Cas12aDS: A visual and fast molecular diagnostics platform based on RPA-CRISPR-Cas12a method for infectious bursal disease virus detection.

Infectious bursal disease (IBD), a major disease of birds, is caused by infectious bursal disease virus (IBDV). The disease can lead to immunosuppression, resulting in huge economic losses in the poultry industry. A specific, rapid, and simple detection method is important for the early diagnosis and prevention and control of IBDV. In this study, we established a naked-eye visual IBDV detection method, named "RPA-Cas12aDS", by combining recombinase polymerase amplification (RPA) with CRISPR-Cas12a-based nucleic acid detection. The detection process can be accomplished in 50 min, and uncapping contamination can be avoided. The detection results can be observed under blue or UV light. We used the RPA-Cas12aDS method to detect IBDV in bursa of Fabricius tissue samples of chickens, and the results were consistent with those obtained using commercial RT-PCR kits. This method presents great potential for visual, rapid, and point-of-care molecular diagnostics of IBDV in poultry.

Rapid detection and tracking of Omicron variant of SARS-CoV-2 using CRISPR-Cas12a-based assay.

BACKGROUND: The newly emerged SARS-CoV-2 variant of concern (VOC) Omicron is spreading quickly worldwide, which manifests an urgent need of simple and rapid assay to detect and diagnose Omicron infection and track its spread. METHODS: To design allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of Omicron variant, and to develop a CRISPR-Cas12a-based assay to specifically detect Omicron variant. RESULTS: Our system showed a low limit of detection of 2 copies per reaction for the plasmid DNA of Omicron variant, and could readily detect Omicron variant in 5 laboratory-confirmed clinical samples and distinguish them from 57 SARS-CoV-2 positive clinical samples (4 virus isolates and 53 oropharyngeal swab specimens) infected with wild-type (N = 8) and the variants of Alpha (N = 17), Beta (N = 17) and Delta (N = 15). The testing results could be measured by fluorescent detector or judged by naked eyes. In addition, no cross-reaction was observed when detecting 16 clinical samples infected with 9 common respiratory pathogens. CONCLUSIONS: The rapid assay could be easily set up in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests and implemented routinely in resource-limited settings to monitor and track the spread of Omicron variant.

CRISPR/Cas12a-Derived electrochemical aptasensor for ultrasensitive detection of COVID-19 nucleocapsid protein.

Fast, affordable, portable, and sensitive technology to detect COVID-19 is critical to address the current outbreak. Here, we present a CRISPR/Cas12a-derived electrochemical aptasensor for cost-effective, fast, and ultrasensitive COVID-19 nucleocapsid protein (Np) detection. First, an electrochemical sensing interface was fabricated by immobilizing methylene blue labeled poly adenines DNA sequence (polyA-MB electrochemical reporter) on a gold electrode surface. Second, an arched probe was prepared via hybridization of Np aptamer and an activator strand. In the presence of COVID-19 Np, the activator strand could be released from the arched probe due to the specific interaction between the target and the aptamer, which then activated the trans-cleavage activity of the CRISPR/Cas12a system. Subsequently, the polyA-MB reporters were cleaved from the electrode surface, decreasing the current of differential pulse voltammetry (DPV) at a potential of -0.27 V(vs. Ag/AgCl). The CRISPR/Cas12a-derived electrochemical aptasensor shows a highly efficient performance for COVID-19 Np detection in 50 pg mL-1 to 100 ng mL-1 with a limit of detection (LOD) low to 16.5 pg mL-1. Notably, the whole process of one test can be completed within 30 min. Simultaneously, the aptasensor displays a high selectivity to other proteins. The further measurements demonstrate that the aptasensor is robust in a natural system for point-of-care testing, such as in tap water, milk, or serum. The aptasensor is universal and expandable and holds great potential in the COVID-19 early diagnosis, environmental surveillance, food security, and other aspects.

Accelerated CRISPR/Cas12a-based small molecule detection using bivalent aptamer.

CRISPR/Cas holds great promise for biosensing applications, however, restricted to nucleic acid targets. Here, we broaden the sensing target of CRISPR/Cas to small molecules via integrating a bivalent aptamer as a recognition component. Using adenosine 5'-triphosphate (ATP) as a model molecule, we found that a bivalent aptamer we selected could shorten the binding time between the aptamer and ATP from 30 min to 3 min, thus dramatically accelerating the detection of ATP. The accelerated bivalent aptamer binding to ATP was mainly ascribed to the extended conformation of the aptamer, which was stabilized through linking with a 5 T bases connector on specific loops of the monovalent aptamer. To facilitate on-site detection, we integrated lateral flow assay (LFA) with the CRISPR/Cas sensing strategy (termed BA-CASLFA) to serve as a visual readout of the presence of ATP. In addition, in the CASLFA platform, due to the unique characteristics of LFA, the thermal step of Cas12a inactivation can be omitted. The BA-CASLFA could output a colorimetric "TURN ON" signal for ATP within 26 min, which could be easily discriminated by the naked eye and sensitively quantified by the portable reader. Furthermore, we showed the versatility of BA-CASLFA for detecting kanamycin using a kanamycin bivalent aptamer obtained through the same design as the ATP bivalent aptamer. Therefore, this strategy is amenable to serve as a general sensing strategy for small molecular targets. The above work opened a new way in developing CRISPR-based on-site sensors for clinic diagnosis, food safety, and environmental analysis.