An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA.

Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a "sample-in-answer-out" single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.

Rapid and Easy-Read Porcine Circovirus Type 4 Detection with CRISPR-Cas13a-Based Lateral Flow Strip.

First identified as a new circovirus in Hunan Province in China in 2019, porcine circovirus (PCV4) is now widely detected in other Chinese provinces and South Korea. In recent years, the virus has threatened pig health and operations in the pig industry. Hence, early PCV4 detection and regular surveillance are required to control the spread of infection and prevent collateral damage to the industry. Due to PCV4 being difficult to isolate in vitro, molecular detection methods, such as conventional PCR and real-time PCR, and serological assays are currently the main methods used for the detection of PCV4 infection. However, they are time-consuming, labor-intensive, and complex and require professional personnel. To facilitate rapid pen-side PCV4 diagnoses, we used clustered regularly interspaced short palindromic repeats (CRISPR) and Cas13a technology to develop a quick testing kit. Five recombinase-aided amplification (RPA) primer sets were designed based on the conserved PCV4-Cap gene nucleotide region, which were used to determine several key lateral flow strip (LFD) characteristics (sensitivity, specificity, and accuracy). The results showed that the RPA-Cas13a-LFD reaction could detect PCV4 within 1.5 h in genomic DNA harboring a minimum of a single copy. Furthermore, the assay showed good specificity and absence of cross-reactivity with PCV2, PCV3, or other porcine viruses. When we tested 15 clinical samples, a high accuracy was also recorded. Therefore, we successfully developed a detection assay that was simple, fast, accurate, and suitable for on-site PCV4 testing.

Gold Nanoparticle Enabled Localized Surface Plasmon Resonance on Unique Gold Nanomushroom Structures for On-Chip CRISPR-Cas13a Sensing.

A novel localized surface plasmon resonance (LSPR) system based on the coupling of gold nanomushrooms (AuNMs) and gold nanoparticles (AuNPs) is developed to enable a significant plasmonic resonant shift. The AuNP size, surface chemistry, and concentration are characterized to maximize the LSPR effect. A 31 nm redshift is achieved when the AuNMs are saturated by the AuNPs. This giant redshift also increases the full width of the spectrum and is explained by the 3D finite-difference time-domain (FDTD) calculation. In addition, this LSPR substrate is packaged in a microfluidic cell and integrated with a CRISPR-Cas13a RNA detection assay for the detection of the SARS-CoV-2 RNA targets. Once activated by the target, the AuNPs are cleaved from linker probes and randomly deposited on the AuNM substrate, demonstrating a large redshift. The novel LSPR chip using AuNP as an indicator is simple, specific, isothermal, and label-free; and thus, provides a new opportunity to achieve the next generation multiplexing and sensitive molecular diagnostic system.

High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple with stabilized graphene field-effect transistor.

False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a "big subtraction" signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/µL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.

Visual detection and differentiation of porcine epidemic diarrhea virus wild-type strains and attenuated vaccine strains using CRISPR/Cas13a-based lateral flow strip.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets. Infections result in high mortality and serious economic losses to the swine industry. PEDV attenuated vaccine does not completely protect against all mutant wild-type strains, and PEDV infection can periodically occur. A sensitive, accurate, and simple detection method for PEDV is needed to reduce the occurrence of the disease. In this study, the CRISPR/Cas13a system was combined with recombinase aided amplification to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The method is based on isothermal detection at 37°C. The results are used for visual readout. The assay had high sensitivity and specificity, with a detection limit of 101 copies/µL for the gene of interest, and no cross-reactivity with other pathogens. The Cas13a detection worked well with clinical samples. This visual, sensitive, and specific nucleic acid detection method based on CRISPR/Cas13a should be a powerful tool for detecting PEDV.

CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter.

Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2',3'-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.

Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR‐based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification‐free nucleic acid detection via harnessing the trans‐cleavage mechanism of Cas13a and ultrasensitive graphene field‐effect transistors (gFETs). CRISPR Cas13a‐gFET achieves the detection of SARS‐CoV‐2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS‐CoV‐2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a‐gFET among the most sensitive amplification‐free nucleic acid diagnostic platforms to date.

Rücktitelbild: Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

Der Nachweis von Nukleinsäuren spielt eine wichtige Rolle in der medizinischen Diagnostik, der Umweltüberwachung und der Lebensmittelsicherheit. In ihrem Forschungsartikel (e202203826) entwickelten Xue Gao, Yi Zhang und Mitarbeiter einen neuen Biosensor für den amplifikationsfreien Nukleinsäurenachweis, indem sie den trans‐Spaltungsmechanismus von Cas13a und ultrasensitive Graphen‐Feldeffekttransistoren (gFETs) nutzten. Die Abbildung zeigt die Cas13a‐vermittelte RNA‐trans‐Spaltung auf der gFET‐Oberfläche für die Sensorsignalübertragung.

Highly Sensitive Detection Method for HV69-70del in SARS-CoV-2 Alpha and Omicron Variants Based on CRISPR/Cas13a.

As SARS-CoV-2 variants continue to evolve, identifying variants with adaptive diagnostic tool is critical to containing the ongoing COVID-19 pandemic. Herein, we establish a highly sensitive and portable on-site detection method for the HV69-70del which exist in SARS-CoV-2 Alpha and Omicron variants using a PCR-based CRISPR/Cas13a detection system (PCR-CRISPR). The specific crRNA (CRISPR RNA) targeting the HV69-70del is screened using the fluorescence-based CRISPR assay, and the sensitivity and specificity of this method are evaluated using diluted nucleic acids of SARS-CoV-2 variants and other pathogens. The results show that the PCR-CRISPR detection method can detect 1 copies/µL SARS-CoV-2 HV69-70del mutant RNA and identify 0.1% of mutant RNA in mixed samples, which is more sensitive than the RT-qPCR based commercial SARS-CoV-2 variants detection kits and sanger sequencing. And it has no cross reactivity with ten other pathogens nucleic acids. Additionally, by combined with our previously developed ERASE (Easy-Readout and Sensitive Enhanced) lateral flow strip suitable for CRISPR detection, we provide a novel diagnosis tool to identify SARS-CoV-2 variants in primary and resource-limited medical institutions without professional and expensive fluorescent detector.

COVID-19 Diagnostics: A Panoramic View on Its Present Scenario, Challenges and Solutions.

The initial strategy to curb the surge of novel coronavirus disease, COVID-19, is prevention and quarantine, which are dependent on early diagnosis. The latest commercial diagnostic methods include AI/ML-based imaging methods and laboratory diagnosis, which differ in their efficiency. The former requires lung imaging and is useful for last stage patients. It was ensured to overcome the limitation of availability of laboratory-based kits, while the latter involves the collection of the suitable sample from an individual (blood sample, nasal or oral swab). Laboratory methods include methods like RT-PCR which is contemporarily contemplated as the benchmark for its quick and efficient SARS-CoV-2 infection detection. Other diagnosis alternatives include Serum Viral Neutralization (SVN) assays involving antigen-antibody reaction with much lower efficiency contrasted to RT-PCR. Apart from these methods, early detection has been key to the treatment of COVID-19, but the lack of sensitive assays to detect low viral titers acts as an impediment. This review presents an overview of detecting COVID-19 with the aid of several diagnostic techniques along with their benefits and limitations.

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2.

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.

Gold Nanoparticle-Labeled CRISPR-Cas13a Assay for the Sensitive Solid-State Nanopore Molecular Counting.

A gold nanoparticle (AuNP) labeled CRISPR-Cas13a nucleic acid assay has been developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, our system utilizes non-covalent conjugates of AuNPs and RNA targets. Upon CRISPR activation, the AuNPs are liberated from the RNA, isolated, and passed through a nanopore sensor. Detection of the AuNPs can be observed as increasing ionic current in the chip. Each AuNP that is detected is enumerated as an event, leading to quantitative of molecular targets. Leveraging the high signal-to-noise ratio enabled by the AuNPs, a detection limit of 50 fM before front-end target amplification is achieved using SARS-CoV-2 RNA segments as a Cas13 target. Furthermore, a dynamic range of six orders of magnitude is demonstrated for quantitative RNA sensing. This simplified AuNP-based CRISPR assay is performed at the physiological temperature without relying on thermal cyclers. In addition, the nanopore reader is similar in size to a smartphone, making the assay system suitable for rapid and portable nucleic acid biomarker detection in either low-resource settings or hospitals.

SARS-CoV-2 detection using quantum dot fluorescence immunochromatography combined with isothermal amplification and CRISPR/Cas13a.

The development of reliable, sensitive, and fast devices for the diagnosis of COVID-19 is of great importance in the pandemic of the new coronavirus. Here, we proposed a new principle of analysis based on a combination of reverse transcription and isothermal amplification of a fragment of the gene encoding the S protein of the SARS-CoV-2 and the CRISPR/Cas13a reaction for cleavage of the specific probe. As a result, the destroyed probe cannot be detected on an immunochromatographic strip using quantum fluorescent dots. Besides, the results can be obtained by an available and inexpensive portable device. By detecting SARS-CoV-2 negative (n = 25) and positive (n = 62) clinical samples including throat swabs, sputum and anal swabs, the assay showed good sensitivity and specificity of the method and could be completed within 1 h without complicated operation and expensive equipment. These superiorities showed its potential for fast point-of-care screening of SARS-CoV-2 during the outbreak, especially in remote and underdeveloped areas with limited equipment and resources.

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics.

The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/µL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

Electrochemical biosensor for nucleic acid amplification-free and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA via CRISPR/Cas13a trans-cleavage reaction.

The outbreak of the COVID-19 pandemic has led to millions of fatalities worldwide. For preventing epidemic transmission, rapid and accurate virus detection methods to early identify infected people are urgently needed in the current situation. Therefore, an electrochemical biosensor based on the trans-cleavage activity of CRISPR/Cas13a was developed in this study for rapid, sensitive, and nucleic-acid-amplification-free detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a redox probe conjugated with ssRNA is immobilized on the electrode surface modified with a nanocomposite (NC) and gold nanoflower (AuNF) for enhancing the sensing performance. The SARS-CoV-2 RNA is captured by the Cas13a-crRNA complex, which triggers the RNase function of Cas13a. The enzymatically activated Cas13a-crRNA complex is subsequently introduced to the reRNA-conjugated electrochemical sensor, and consequently cleaves the reRNA. A change in current occurs due to the release of the redox molecule labeled on the reRNA, which is trans-cleaved from the Cas13a-crRNA complex. The biosensor can detect as low as 4.4 × 10-2 fg/mL and 8.1 × 10-2 fg/mL of ORF and S genes, respectively, over a wide dynamic range (1.0 × 10-1 to 1.0 × 105 fg/mL). Moreover, the biosensor was evaluated by measuring SARS-CoV-2 RNA spiked in artificial saliva. The recovery of the developed sensor was found to be in an agreeable range of 96.54-101.21%. The designed biosensor lays the groundwork for pre-amplification-free detection of ultra-low concentrations of SARS-CoV-2 RNA and on-site and rapid diagnostic testing for COVID-19.

A CRISPR-based and post-amplification coupled SARS-CoV-2 detection with a portable evanescent wave biosensor.

The continuing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has spread globally and its reliable diagnosis is one of the foremost priorities for protecting public health. Herein a rapid (<1 h), easy-to-implement, and accurate CRISPR-based evanescent wave fluorescence biosensing platform for detection of SARS-CoV-2 is reported. The collateral effect of Cas13a is combined with a universal autonomous enzyme-free hybridization chain reaction (HCR) by designing a cleavage hairpin reporter, which is cleaved upon target recognition, and hence releasing the initiator sequence to trigger the downstream HCR circuits. Detection of HCR assemblies is accomplished by first adsorbing to the desthiobiotin-modified optical fiber, followed by fluorescence emission induced by an evanescent field. Three Cas13a crRNAs targeting the genes of S, N and Orf1ab of SARS-CoV-2 are programmed to specifically target SARS-CoV-2 or broadly detect related coronavirus strains, such as MERS-CoV and SARS-CoV. The HCR amplification coupled Cas13a-based biosensing platform is capable of rapid detection of SARS-CoV-2 with attomolar sensitivity. This method is further validated by adding target RNA of SARS-CoV-2 in negative oropharyngeal swabs. The good discrimination capability of this technique demonstrates its promising potential for point-of-care diagnosis of COVID-19.

A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection.

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.

Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.