Digital CRISPR Systems for the Next Generation of Nucleic Acid Quantification.

Digital CRISPR (dCRISPR) assays are an emerging platform of molecular diagnostics. Digital platforms introduce absolute quantification and increased sensitivity to bulk CRISPR assays. With ultra-specific targeting, isothermal operation, and rapid detection, dCRISPR systems are well-prepared to lead the field of molecular diagnostics. Here we summarized the common Cas proteins used in CRISPR detection assays. The methods of digital detection and critical performance factors are examined. We formed three strategies to frame the landscape of dCRISPR systems: (1) amplification free, (2) in-partition amplification, and (3) two-stage amplification. We also compared the performance of all systems through the limit of detection (LOD), testing time, and figure of merit (FOM). This work summarizes the details of digital CRISPR platforms to guide future development. We envision that improvements to LOD and dynamic range will position dCRISPR as the leading platform for the next generation of molecular biosensing.

Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.

Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.

CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes significant damage to pine trees and, thus, poses a serious threat to pine forests worldwide, particularly in China, Korea, and Japan. A fast, affordable, and ultrasensitive detection of B. xylophilus is urgently needed for disease diagnosis. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have reshaped molecular diagnosis, with high speed, precision, specificity, strength, efficiency, and versatility. Herein, we established two isothermal diagnostics methods based on CRISPR-based platforms (CRISPR/Cas12a and CRISPR/Cas13a) for B. xylophilus-specific detection via fluorescence or lateral-flow strip readout. The guide RNA and CRISPR RNA were designed to target the 5S ribosomal DNA intergenic spacer sequences region of B. xylophilus. Recombinase-aided amplification was used for preamplification whose reaction condition was 37°C for 15 min. The sensitivity of CRISPR/Cas12a could reach 94 copies/µl of plasmid DNA, or 2.37 copies/µl of purified genomic DNA (gDNA) within 45 min at 37°C, while the sensitivity of CRISPR/Cas13a was 1,000 times higher than that of CRISPR/Cas12a of plasmid DNA in 15 min or 100 times higher of purified gDNA at the minimum reaction time of 4 min via fluorescence measurement. The CRISPR/Cas12a assay enabled the detection of 0.01 PWNs per 100 mg of pine wood, 10 times higher than that of the CRISPR/Cas13a assay. This work enriches molecular detection approaches for B. xylophilus and provides huge potential for ultrasensitive and rapid methods to detect B. xylophilus in pine wood, facilitating point-of-sample diagnostic processing for pine wilt disease management.

Engineered crRNA for CRISPR/Cas-assisted biosensing.

CRISPR/Cas-based diagnostics (CRISPR-Dx) face challenges, including difficulty in detecting ultrashort nucleotides, preamplification dependency, cross-contamination, insufficiency in on-pot detection paradigms, and inconvenience in detecting non-nucleic acid targets. This forum outlines the advances in engineered CRISPR RNA (crRNA) that address the aforementioned problems, highlighting challenges, opportunities, and future directions.

Scanning amplicons with CRISPR-Dx detects endangered amphibians in environmental DNA.

More efficient methods for extensive biodiversity monitoring are required to support rapid measures to address the biodiversity crisis. While environmental DNA (eDNA) metabarcoding and quantitative PCR (qPCR) methods offer advantages over traditional monitoring approaches, their large-scale application is limited by the time and labour required for developing assays and/or for analysis. CRISPR (clustered regularly interspaced short palindromic repeats) diagnostic technologies (Dx) may overcome some of these limitations, but they have been used solely with species-specific primers, restricting their versatility for biodiversity monitoring. Here, we demonstrate the feasibility of designing species-specific CRISPR-Dx assays in silico within a short metabarcoding fragment using a general primer set, a methodology we term 'ampliscanning', for 18 of the 22 amphibian species in Switzerland. We sub-selected nine species, including three classified as regionally endangered, to test the methodology using eDNA sampled from ponds at nine sites. We compared the ampliscanning detections to data from traditional monitoring at these sites. Ampliscanning was successful at detecting target species with different prevalences across the landscape. With only one visit, we detected more species per site than three traditional monitoring visits (visual and acoustic detections by trained experts), in particular more elusive species and previously undocumented but expected populations. Ampliscanning detected 25 species/site combinations compared to 12 with traditional monitoring. Sensitivity analyses showed that larger numbers of field visits and PCR replicates are more important for reliable detection than many technical replicates at the CRISPR-Dx assay level. Given the reduced sampling and analysis effort, our results highlight the benefits of eDNA and CRISPR-Dx combined with universal primers for large-scale monitoring of multiple endangered species across landscapes to inform conservation measures.

SynNotch-Programmed Macrophages for Cancerous Cell Detection and Sensing.

Synthetic Notch (synNotch) receptors have enabled mammalian cells to sense extracellular ligands and respond by activating user-prescribed transcriptional programs. Based on the synNotch system, we describe a cell-based in vivo sensor for cancerous cell detection. We attempted to engineer synNotch-programmed macrophages to sense cancer cells via urinary analysis of human chorionic gonadotropin (HCGB5). Principally, when the synNotch receptors of macrophages bind to the ligands of cancer cells, Notch is activated and undergoes intramembrane proteolysis to release the transcriptional activator into the nucleus. The transcriptional activator targets and activates downstream gene expression, such as human chorionic gonadotropin (HCGB5) in macrophages. When HCGB5 is secreted extracellularly into urine, it can be detected with commercial HCGB5 colloidal gold test strips. As a proof of principle, we demonstrated the feasibility of synNotch-programmed macrophages in detecting breast cancer cells engineered with artificial EGFP ligands. We demonstrated that HCGB5 expression was only induced when the cancer cell expressing EGFP ligands is present; thereby, extracellular HCGB5 expression is directly proportional to the number of cancer cells. Further optimizations of the synNotch system can realize the ultimate goal of establishing cell-based in vivo sensors as the paragon of cancer diagnostics for point-of-care testing and home self-test.

Sensitive Small Molecule Aptasensing based on Hybridization Chain Reaction and CRISPR/Cas12a Using a Portable 3D-Printed Visualizer.

Next-generation biosensing tools based on CRISPR/Cas have revolutionized the molecular detection. A number of CRISPR/Cas-based biosensors have been reported for the detection of nucleic acid targets. The establishment of efficient methods for non-nucleic acid target detection would further broaden the scope of this technique, but up to now, the concerning research is limited. In the current study, we reported a versatile biosensing platform for non-nucleic acid small-molecule detection called SMART-Cas12a (small-molecule aptamer regulated test using CRISPR/Cas12a). Simply, hybridization chain reaction cascade signal amplification was first trigged by functional nucleic acid (aptamer) through target binding. Then, the CRISPR/Cas system was integrated to recognize the amplified products followed by activation of the trans-cleavage. As such, the target can be ingeniously converted to nucleic acid signals and then fluorescent signals that can be readily visualized and analyzed by a customized 3D-printed visualizer with the help of a home-made App-enabled smartphone. Adenosine triphosphate was selected as a model target, and under the optimized conditions, we achieved fine analytical performance with a linear range from 0.1 to 750 µM and a detection limit of 1.0 nM. The satisfactory selectivity and recoveries that we have obtained further demonstrated this method to be suitable for a complex sample environment. The sample-to-answer time was less than 100 min. Our work not only expanded the reach of the CRISPR-Cas system in biosensing but also provided a prototype method that can be generalized for detecting a wider range of analytes with desirable adaptability, sensitivity, specificity, and on-site capability.

A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel.

The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.

Capturing nucleic acid variants with precision using CRISPR diagnostics.

CRISPR/Cas systems have the ability to precisely target nucleotide sequences and enable their rapid identification and modification. While nucleotide modification has enabled the therapeutic correction of diseases, the process of identifying the target DNA or RNA has greatly expanded the field of molecular diagnostics in recent times. CRISPR-based DNA/RNA detection through programmable nucleic acid binding or cleavage has been demonstrated for a large number of pathogenic and non-pathogenic targets. Combining CRISPR detection with nucleic acid amplification and a terminal signal readout step allowed the development of numerous rapid and robust nucleic acid platforms. Wherever the Cas effector can faithfully distinguish nucleobase variants in the target, the platform can also be extended for sequencing-free rapid variant detection. Some initial PAM disruption-based SNV detection reports were limited to finding or integrating mutated/mismatched nucleotides within the PAM sequences. In this review, we try to summarize the developments made in CRISPR diagnostics (CRISPRDx) to date emphasizing CRISPR-based SNV detection. We also discuss the applications where such diagnostic modalities can be put to use, covering various fields of clinical research, SNV screens, disease genotyping, primary surveillance during microbial infections, agriculture, food safety, and industrial biotechnology. The ease of rapid design and implementation of such multiplexable assays can potentially expand the applications of CRISPRDx in the domain of affinity-based target sequencing, with immense possibilities for low-cost, quick, and widespread usage. In the end, in combination with proximity assays and a suicidal gene approach, CRISPR-based in vivo SNV detection and cancer cell targeting can be formulated as personalized gene therapy.

Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review.

Various non-nucleic acid targets (ions, small molecules, polysaccharides, peptides/proteins/enzymes, cells, transcription factors) are important biomarkers. They play important roles in environmental protection, food safety and medical diagnosis. Therefore, it is necessary to detect non-nucleic acid targets from various samples before the situation deteriorates. Derived from prokaryotic immune systems, CRISPR/Cas tools have exhibited great promise in the field of biosensing, in addition to the well-known gene-editing function. However, most reported CRISPR/Cas-based biosensors are for nucleic acid detection and the application of non-nucleic acid targets is still in its infancy. To fully explore the potential of CRISPR/Cas-based biosensing systems, it is of great significance to summarize the strategies and prospects of CRISPR/Cas toolboxes in non-nucleic acid targets recognition. In this review, we introduced CRISPR/Cas systems and their characteristics in the field of detection. The progress of detecting six non-nucleic acid targets was outlined and reviewed based on CRISPR/Cas systems coupled with biotransduction elements, including aptamers, DNAzymes, riboswitches, enzymatic reactions, transcription factors, antigen-antibody interactions, allosteric probes, in vitro transcription processes, steric hindrance effectors, etc. The development challenges and prospects in this field were also put forward. As such, this comprehensive review would provide valuable information for the expansion of the powerful CRISPR/Cas toolboxes into multiple detection fields.

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR-Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR-Cas-based nucleic acid detection strategies and the application of CRISPR-Cas as a potential antiviral agent in the context of COVID-19.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip.

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.

SARS-CoV-2, the virus responsible for COVID-19, has a genome made of RNA (a nucleic acid similar to DNA) that can mutate, potentially making the disease more transmissible, and more lethal. Most countries have monitored the rise of mutated strains using a technique called next generation sequencing (NGS), which is time-consuming, expensive and requires skilled personnel. Sometimes the mutations to the virus are so small that they can only be detected using NGS. Finding cheaper, simpler and faster SARS-CoV-2 tests that can reliably detect mutated forms of the virus is crucial for public health authorities to monitor and manage the spread of the virus. Lateral flow tests (the same technology used in many pregnancy tests) are typically cheap, fast and simple to use. Typically, lateral flow assay strips have a band of immobilised antibodies that bind to a specific protein (or antigen). If a sample contains antigen molecules, these will bind to the immobilised antibodies, causing a chemical reaction that changes the colour of the strip and giving a positive result. However, lateral flow tests that use antibodies cannot easily detect nucleic acids, such as DNA or RNA, let alone mutations in them. To overcome this limitation, lateral flow assays can be used to detect a protein called Cas9, which, in turn, is able to bind to nucleic acids with specific sequences. Small changes in the target sequence change how well Cas9 binds to it, meaning that, in theory, this approach could be used to detect small mutations in the SARS-CoV-2 virus. Kumar et al. made a lateral flow test that could detect a Cas9 protein that binds to a nucleic acid sequence found in a specific mutant strain of SARS-CoV-2. This Cas9 was highly sensitive to changes in its target sequence, so a small mutation in the target nucleic acid led to the protein binding less strongly, and the signal from the lateral flow test being lost. This meant that the lateral flow test designed by Kumar et al. could detect mutations in the SARS-CoV-2 virus at a fraction of the price of NGS approaches if used only for diagnosis. The lateral flow test was capable of detecting mutant viruses in patient samples too, generating a colour signal within an hour of a positive sample being run through the assay. The test developed by Kumar et al. could offer public health authorities a quick and cheap method to monitor the spread of mutant SARS-CoV-2 strains; as well as a way to determine vaccine efficacy against new strains.

iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.

Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.