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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.
Drug Des Devel Ther ; 16: 951-972, 2022.
Article in English | MEDLINE | ID: covidwho-1779832

ABSTRACT

The COVID-19 has plunged the world into a pandemic that affected millions. The continually emerging new variants of concern raise the question as to whether the existing vaccines will continue to provide sufficient protection for individuals from SARS-CoV-2 during natural infection. This narrative review aims to briefly outline various immunotherapeutic options and discuss the potential of clustered regularly interspaced short palindromic repeat (CRISPR Cas system technology against COVID-19 treatment as specific cure. As the development of vaccine, convalescent plasma, neutralizing antibodies are based on the understanding of human immune responses against SARS-CoV-2, boosting human body immune responses in case of SARS-CoV-2 infection, immunotherapeutics seem feasible as specific cure against COVID-19 if the present challenges are overcome. In cell based therapeutics, apart from the high costs, risks and side effects, there are technical problems such as the production of sufficient potent immune cells and antibodies under limited time to treat the COVID-19 patients in mild conditions prior to progression into a more severe case. The CRISPR Cas technology could be utilized to refine the specificity and safety of CAR-T cells, CAR-NK cells and neutralizing antibodies against SARS-CoV-2 during various stages of the COVID-19 disease progression in infected individuals. Moreover, CRISPR Cas technology are proposed in hypotheses to degrade the viral RNA in order to terminate the infection caused by SARS-CoV-2. Thus personalized cocktails of immunotherapeutics and CRISPR Cas systems against COVID-19 as a strategy might prevent further disease progression and circumvent immunity escape.


Subject(s)
COVID-19 , CRISPR-Cas Systems , Antibodies, Neutralizing , COVID-19/drug therapy , COVID-19/therapy , Disease Progression , Humans , Immunization, Passive , Immunologic Factors , SARS-CoV-2
3.
Biosensors (Basel) ; 12(3)2022 Mar 02.
Article in English | MEDLINE | ID: covidwho-1760369

ABSTRACT

Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.


Subject(s)
Campylobacter jejuni , CRISPR-Cas Systems , Magnetic Phenomena , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods
4.
Microb Pathog ; 165: 105498, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1757669

ABSTRACT

OBJECTIVE: To estimate the accuracy of clustered regularly interspaced short palindromic repeats (CRISPR) in determining coronavirus disease-19 (COVID-19). METHODS: As of January 31, 2022, PubMed, Web of Science, Embase, Science Direct, Wiley and Springer Link were searched. Sensitivity, specificity, likelihood ratio (LR), diagnostic odds ratio (DOR) and area under the summary receiver-operating characteristic (AUC) curve were used to assess the accuracy of CRISPR. RESULTS: According to the inclusion criteria, 5857 patients from 54 studies were included in this meta-analysis. The pooled sensitivity, specificity and AUC were 0.98, 1.00 and 1.00, respectively. For CRISPR-associated (Cas) proteins-12, the sensitivity, specificity was 0.96, 1.00, respectively. For Cas-13, the sensitivity and specificity were 0.99 and 0.99. CONCLUSION: This meta-analysis showed that the diagnostic performance of CRISPR is close to the gold standard, and it is expected to meet the Point of care requirements in resource poor areas.


Subject(s)
COVID-19 , COVID-19/diagnosis , CRISPR-Cas Systems , Humans
5.
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
6.
Nat Biomed Eng ; 6(3): 286-297, 2022 03.
Article in English | MEDLINE | ID: covidwho-1751719

ABSTRACT

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.


Subject(s)
COVID-19 , RNA, Viral , COVID-19/diagnosis , CRISPR-Cas Systems , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
7.
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
8.
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
9.
Cell Rep ; 38(10): 110476, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1729612

ABSTRACT

Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.


Subject(s)
Bacterial Toxins , COVID-19 , Bacterial Toxins/genetics , CRISPR-Cas Systems , Gene Editing , Humans , RNA, Guide/metabolism , SARS-CoV-2
10.
Viruses ; 14(2)2022 01 18.
Article in English | MEDLINE | ID: covidwho-1715743

ABSTRACT

The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 101 copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.


Subject(s)
African Swine Fever Virus/isolation & purification , African Swine Fever/diagnosis , CRISPR-Cas Systems , African Swine Fever/virology , African Swine Fever Virus/genetics , Animals , Genotype , Reagent Strips , Recombinases/genetics , Recombinases/metabolism , Sensitivity and Specificity , Sus scrofa , Swine , Time Factors
11.
Biosensors (Basel) ; 12(2)2022 Jan 19.
Article in English | MEDLINE | ID: covidwho-1715104

ABSTRACT

CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.


Subject(s)
CRISPR-Cas Systems , RNA
12.
Curr Protoc ; 2(2): e385, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1709165

ABSTRACT

The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets.


Subject(s)
COVID-19 , SARS-CoV-2 , CRISPR-Cas Systems , Humans , Nucleic Acid Amplification Techniques , Pandemics
13.
Biomed Pharmacother ; 148: 112743, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1703121

ABSTRACT

Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.


Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Genetic Therapy/methods , Virus Diseases/therapy , COVID-19/therapy , Genome, Viral , HIV Infections/therapy , Hepatitis B/therapy , Herpesviridae Infections/therapy , Humans , Papillomavirus Infections/therapy , SARS-CoV-2
14.
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
15.
Viruses ; 14(2)2022 02 14.
Article in English | MEDLINE | ID: covidwho-1687053

ABSTRACT

The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics.


Subject(s)
Computer Simulation , Genome, Viral , RNA, Viral/genetics , SARS-CoV-2/genetics , CRISPR-Cas Systems , Humans , Spike Glycoprotein, Coronavirus/genetics
16.
Molecules ; 27(3)2022 Feb 07.
Article in English | MEDLINE | ID: covidwho-1686901

ABSTRACT

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is best known for its role in genomic editing. It has also demonstrated great potential in nucleic acid biosensing. However, the specificity limitation in CRISPR/Cas has created a hurdle for its advancement. More recently, nucleic acid aptamers known for their high affinity and specificity properties for their targets have been integrated into CRISPR/Cas systems. This review article gives a brief overview of the aptamer and CRISPR/Cas technology and provides an updated summary and discussion on how the two distinctive nucleic acid technologies are being integrated into modern diagnostic and therapeutic applications.


Subject(s)
Aptamers, Nucleotide/therapeutic use , Biosensing Techniques/methods , CRISPR-Cas Systems , Gene Editing , Precision Medicine , Humans
17.
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
18.
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
19.
J Nanobiotechnology ; 20(1): 41, 2022 Jan 21.
Article in English | MEDLINE | ID: covidwho-1643157

ABSTRACT

Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.


Subject(s)
CRISPR-Cas Systems/physiology , DNA, Viral/isolation & purification , Nucleic Acid Amplification Techniques , Animals , Biosensing Techniques/methods , Biosensing Techniques/trends , COVID-19/virology , DNA, Viral/analysis , Environmental Pollutants/analysis , Environmental Pollutants/isolation & purification , Food Contamination/analysis , Humans , Molecular Typing/methods , Molecular Typing/trends , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/trends , SARS-CoV-2/genetics , Virology/methods , Virology/trends , Virus Diseases/classification , Virus Diseases/diagnosis , Virus Diseases/virology
20.
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
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