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1.
Nat Commun ; 13(1): 1937, 2022 Apr 11.
Article in English | MEDLINE | ID: covidwho-1783981

ABSTRACT

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.


Subject(s)
Biosensing Techniques , COVID-19 , CRISPR-Cas Systems/genetics , Humans , RNA, Guide/genetics , RNA, Guide/metabolism , RNA, Viral/genetics , SARS-CoV-2/genetics
2.
Chem Commun (Camb) ; 58(28): 4484-4487, 2022 Apr 05.
Article in English | MEDLINE | ID: covidwho-1751771

ABSTRACT

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a primer exchange reaction (PER). By ingeniously integrating the PER cascade and CRISPR/cas12a system, this method can achieve convenient detection of the target RNA in 40 min and distinguish a single-base mutation from the target sequence, demonstrating its superior analytical performance.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics
3.
Cells ; 11(6)2022 03 15.
Article in English | MEDLINE | ID: covidwho-1742343

ABSTRACT

Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.


Subject(s)
COVID-19 , Virus Diseases , Viruses , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , DNA Viruses , Host Microbial Interactions , Humans , SARS-CoV-2/genetics , Virus Diseases/diagnosis , Virus Diseases/genetics , Viruses/genetics
4.
Talanta ; 243: 123388, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1735000

ABSTRACT

Nucleic acid detection technology is now widely used in scientific research and clinical testing, such as infectious and genetic diseases screening, molecular diagnosis of tumors and pharmacogenomic research, which is also an important part of in vitro diagnostics (IVD). However, with the increasing requirements of diagnosis and treatment, existing nucleic acid detection technologies are facing challenges in dealing with the current problems (especially since the outbreak of coronavirus disease in 2019 (Covid-19)). Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas)-based diagnostics have become a hot spot of attention. CRISPR/Cas has been developed as a molecular detection tool besides scientific research in biology and medicine fields, and some CRISPR-based products have already been translated. It is known as the "next-generation molecular diagnostic technology" because of its advantages such as easy design and accurate identification. CRISPR/Cas relies on pre-amplification of target sequences and subsequent detection of Cas proteins. Combining the CRISPR/Cas system with various isothermal nucleic acid amplification strategies can generate amplified detection signals, enrich low abundance molecular targets, improve the specificity and sensitivity of analysis, and develop point-of-care (POC) diagnostic techniques. In this review, we analyze the current status of CRISPR/Cas systems and isothermal amplification, report the advantages of combining the two and summarize the recent progress with the integration of both technologies with POC sensors in the nucleic acid field. In addition, the challenges and future prospects of CRISPR technology combined with isothermal amplification strategies in biosensing and clinical applications are discussed.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques , Point-of-Care Systems
5.
Biosens Bioelectron ; 205: 114098, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1693895

ABSTRACT

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.


Subject(s)
Biosensing Techniques , COVID-19 , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , SARS-CoV-2/genetics
6.
Bioessays ; 44(4): e2100286, 2022 04.
Article in English | MEDLINE | ID: covidwho-1680275

ABSTRACT

CRISPR-Cas technology accelerates development of fast, accurate, and portable diagnostic tools, typified by recent applications in COVID-19 diagnosis. Parasitic helminths cause devastating diseases afflicting 1.5 billion people globally, representing a significant public health and economic burden, especially in developing countries. Currently available diagnostic tests for worm infection are neither sufficiently sensitive nor field-friendly for use in low-endemic or resource-poor settings, leading to underestimation of true prevalence rates. Mass drug administration programs are unsustainable long-term, and diagnostic tools - required to be rapid, specific, sensitive, cost-effective, and user-friendly without specialized equipment and expertise - are urgently needed for rapid mapping of helminthic diseases and monitoring control programs. We describe the key features of the CRISPR-Cas12/13 system and emphasise its potential for the development of effective tools for the diagnosis of parasitic and other neglected tropical diseases (NTDs), a key recommendation of the NTDs 2021-2030 roadmap released by the World Health Organization.


Subject(s)
COVID-19 , Parasites , Parasitic Diseases , Animals , COVID-19 Testing , CRISPR-Cas Systems/genetics , Humans , Parasites/genetics
7.
Int J Mol Sci ; 23(3)2022 Feb 03.
Article in English | MEDLINE | ID: covidwho-1674668

ABSTRACT

CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.


Subject(s)
Diagnostic Techniques and Procedures/trends , Disease/genetics , Gene Editing/methods , COVID-19/genetics , CRISPR-Cas Systems/genetics , DNA/genetics , Diagnosis , Humans , Reproducibility of Results , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
8.
Biosens Bioelectron ; 196: 113701, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1638371

ABSTRACT

Although CRISPR-Cas12a and CRISPR-Cas13a systems work individually effective on gene detection, their multiplex detection capability is limited due to the lack of specific probe cleavage mechanism. Herein we present a high-efficient dual-gene diagnostic technique based on the orthogonal DNA/RNA collateral cleavage mechanism of Cas12a/Cas13a system. In this design, dual-gene amplified products from the multiplex recombinase polymerase amplification (RPA) were simultaneously detected by Cas12a and Cas13a assay in a single tube. The resulting orthogonal DNA/RNA collateral cleavage can specifically illuminate two spectral differentiated DNA and RNA probes, respectively. By integrating with the smartphone-based fluorescence readout, a portable detection platform is achieved. As a proof-of-concept, reliable dual-gene detection of SARS-CoV-2 and African Swine fever virus (ASFV) were demonstrated, exhibiting 100% sensitivity and specificity for clinical samples analysis (32 swab specimens for SARS-CoV-2 and 35 ASFV suspected swine blood samples). This developed portable dual-gene detection platform can provide accurate point-of-care screening of infectious diseases in resources-limited settings.


Subject(s)
African Swine Fever Virus , Biosensing Techniques , COVID-19 , Animals , CRISPR-Cas Systems/genetics , Humans , SARS-CoV-2 , Swine
9.
PLoS One ; 16(12): e0261778, 2021.
Article in English | MEDLINE | ID: covidwho-1613357

ABSTRACT

Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.


Subject(s)
COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/genetics , CRISPR-Cas Systems/genetics , DNA Primers/genetics , Diagnostic Tests, Routine/methods , Humans , Mutation/genetics , RNA, Guide/genetics , RNA, Guide/metabolism , Sensitivity and Specificity
10.
Gene ; 818: 146136, 2022 Apr 15.
Article in English | MEDLINE | ID: covidwho-1611737

ABSTRACT

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas protein (CRISPR-Cas) has turned out to be a very important tool for the rapid detection of viruses. This can be used for the identification of the target site in a virus by identifying a 3-6 nt length Protospacer Adjacent Motif (PAM) adjacent to the potential target site, thus motivating us to adopt CRISPR-Cas technique to identify SARS-CoV-2 as well as other members of Coronaviridae family. In this regard, we have developed a fast and effective method using k-mer technique in order to identify the PAM by scanning the whole genome of the respective virus. Subsequently, palindromic sequences adjacent to the PAM locations are identified as the potential target sites. Palindromes are considered in this work as they are known to identify viruses. Once all the palindrome-PAM combinations are identified, PAMs specific for the RNA-guided DNA Cas9/Cas12 endonuclease are identified to bind and cut the target sites. In this regard, PAMs such as 5'-TGG-3' and 5'-TTTA-3' in NSP3 and Exon for SARS-CoV-2, 5'-GGG-3' and 5'-TGG-3' in Exon and NSP2 for MERS-CoV and 5'-AGG-3' and 5'-TTTG-3' in Helicase and NSP3 respectively for SARS-CoV-1 are identified corresponding to SpCas9 and FnCas12a endonucleases. Finally, to recognise the target sites of Coronaviridae family as cleaved by SpCas9 and FnCas12a, complements of the palindromic target regions are designed as primers or guide RNA (gRNA). Therefore, such complementary gRNAs along with respective Cas proteins can be considered in assays for the identification of SARS-CoV-2, MERS-CoV and SARS-CoV-1.


Subject(s)
CRISPR-Cas Systems/genetics , Inverted Repeat Sequences/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Base Sequence , CRISPR-Associated Protein 9/metabolism , Gene Editing , Humans
11.
Med Hypotheses ; 159: 110754, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1586984

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a new respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and now spreads globally. Currently, therapeutics and effective treatment options remain scarce and there is no proven drug to treat COVID-19. Targeting the positive-sense RNA genome and viral mRNAs of SARS-CoV-2 to simultaneously degrade viral genome templates for replication and viral mRNAs for essential gene expression would be a strategy to completely realize virus elimination. Type VI CRISPR enzymes Cas13 have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allows for RNA cleavage and degradation. The precise viral RNA detection and antiviral application of the CRISPR/Cas13 system depend on high-efficient and minimal off-target crRNAs. Although a computer-based algorithm has been applied for the design of crRNAs targeting SRAS-CoV-2, the experimental screening system to identify optimal crRNA is not available. We develop a one-step experimental screening system to identify high-efficient crRNAs with minimal off-target effects for CRISPR/Cas13-based SARS-CoV-2 elimination. This platform provides the foundation for CRISPR/Cas13-based diagnostics and therapeutics for COVID-19. This platform is versatile and could also be applied for crRNAs screening for other RNA viruses.


Subject(s)
COVID-19 , SARS-CoV-2 , CRISPR-Cas Systems/genetics , Genome, Viral , Humans , RNA, Viral
12.
ACS Synth Biol ; 11(1): 317-324, 2022 01 21.
Article in English | MEDLINE | ID: covidwho-1586042

ABSTRACT

Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars ("Bt11" and "MON89034") from nontransgenic corn cultivars ("yellow", "shenyu", "xianyu", and "jingke"). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.


Subject(s)
Biosensing Techniques , G-Quadruplexes , CRISPR-Cas Systems/genetics , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction
13.
Nucleic Acids Res ; 49(22): 13122-13134, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1555464

ABSTRACT

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.


Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems/genetics , Endodeoxyribonucleases/metabolism , Endoribonucleases/metabolism , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , COVID-19 , Cell Line , Chlorocebus aethiops , Humans , Prophages/genetics , Vero Cells , Vibrio/virology
14.
Anal Chem ; 93(48): 16184-16193, 2021 12 07.
Article in English | MEDLINE | ID: covidwho-1531973

ABSTRACT

Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/µL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.


Subject(s)
COVID-19 , SARS-CoV-2 , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques
15.
Chem Commun (Camb) ; 57(92): 12270-12272, 2021 Nov 19.
Article in English | MEDLINE | ID: covidwho-1506302

ABSTRACT

An automated Cas12a-microfluidic system was constructed to distinguish the B.1.617.2 (delta) variant of SARS-CoV-2 from the wild-type virus rapidly and was validated using 30 clinical samples, showing 100% consistency with next-generation sequencing. It will be a potential tool for the rapid differential diagnosis of the delta variant of SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Microfluidics/methods , SARS-CoV-2/genetics , Automation , COVID-19/virology , High-Throughput Nucleotide Sequencing , Humans , Mutation , Polymerase Chain Reaction , RNA, Guide/genetics , RNA, Guide/metabolism , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
16.
Viruses ; 13(10)2021 10 04.
Article in English | MEDLINE | ID: covidwho-1481009

ABSTRACT

The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been repurposed as genome editors to induce targeted double-strand breaks at almost any location in the genome. Thus, CRISPR/Cas genome editors can also be utilized to generate disease-resistant or resilient livestock, develop vaccines, and further understand virus-host interactions. Genes of interest in animals and viruses can be targeted to understand their functions during infection. Furthermore, transgenic animals expressing CRISPR/Cas can be generated to target the viral genome upon infection. Genetically modified livestock can thereby reduce disease outbreaks and decrease zoonotic threats.


Subject(s)
Gene Editing/methods , Livestock/virology , Viruses/genetics , Animal Husbandry/methods , Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Engineering , Host Microbial Interactions/genetics , Virus Diseases/prevention & control , Viruses/pathogenicity
17.
Chem Soc Rev ; 50(21): 11844-11869, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1454829

ABSTRACT

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems have revolutionized biological and biomedical sciences in many ways. The last few years have also seen tremendous interest in deploying the CRISPR-Cas toolbox for analytical and diagnostic assay development because CRISPR-Cas is one of the most powerful classes of molecular machineries for the recognition and manipulation of nucleic acids. In the short period of development, many CRISPR-enabled assays have already established critical roles in clinical diagnostics, biosensing, and bioimaging. We describe in this review the recent advances and design principles of CRISPR mediated analytical tools with an emphasis on the functional roles of CRISPR-Cas machineries as highly efficient binders and molecular scissors. We highlight the diverse engineering approaches for molecularly modifying CRISPR-Cas machineries and for devising better readout platforms. We discuss the potential roles of these new approaches and platforms in enhancing assay sensitivity, specificity, multiplexity, and clinical outcomes. By illustrating the biochemical and analytical processes, we hope this review will help guide the best use of the CRISPR-Cas toolbox in detecting, quantifying and imaging biologically and clinically important molecules and inspire new ideas, technological advances and engineering strategies for addressing real-world challenges such as the on-going COVID-19 pandemic.


Subject(s)
COVID-19 , Nucleic Acids , CRISPR-Cas Systems/genetics , Humans , Pandemics , SARS-CoV-2
18.
Biosensors (Basel) ; 11(10)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1444102

ABSTRACT

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.


Subject(s)
COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Testing/instrumentation , COVID-19 Testing/methods , Humans , Limit of Detection , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
19.
Nat Commun ; 12(1): 5653, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440472

ABSTRACT

Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/µl sensitivity in amplification-free and 60 copies/µl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , SARS-CoV-2/isolation & purification , COVID-19/blood , COVID-19/virology , Humans , Limit of Detection , RNA, Viral/genetics , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
20.
Nano Lett ; 21(19): 8393-8400, 2021 10 13.
Article in English | MEDLINE | ID: covidwho-1428741

ABSTRACT

The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/µL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.


Subject(s)
COVID-19 , Nanopores , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Sensitivity and Specificity
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