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1.
J Infect Dev Ctries ; 16(4): 604-607, 2022 04 30.
Article in English | MEDLINE | ID: covidwho-1841515

ABSTRACT

INTRODUCTION: Duration of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) shedding is important for infection control. The presence of SARS-CoV-2 subgenomic RNA (sgRNA) leader indicates that the virus is replicative. This study examined the shedding duration of SARS-CoV-2 sgRNA leader and genomic RNA (gRNA) in diverse respiratory specimens. METHODOLOGY: One hundred and eleven respiratory specimens collected sequentially from 10 COVID-19 patients with real-time RT-PCR SARS-CoV-2 orf1ab gene confirmed positive admitted to King Chulalongkorn Memorial Hospital were examined for SARS-CoV-2 E sgRNA leader and E gRNA by using Real-time reverse transcription PCR (qRT-PCR). These specimens included nasopharyngeal swab and throat swabs, nasal swab and throat swabs, sputum, and endotracheal aspirate, and were collected from the first day of admission until the time of orf1ab real-time RT-PCR negative of at least 2-4 consecutive days. RESULTS: E sgRNA leader could only be detectable in specimens with ≥ 1E+05 virus E gene copies per ml within the first 15 days after hospitalization. SARS-CoV-2 sgRNA leader was undetectable from one to 15 days earlier than that of gRNA in all patients. Re-shedding of sgRNA was evident in 2 cases, both on a single occasion after being undetectable for 3-10 days. CONCLUSIONS: Assessment of the presence of sgRNA leader may be useful for therapeutic planning.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , RNA, Guide , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics
2.
Viruses ; 14(5)2022 05 05.
Article in English | MEDLINE | ID: covidwho-1820426

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.


Subject(s)
Replicon , SARS-CoV-2 , Antiviral Agents/pharmacology , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Promoter Regions, Genetic , RNA, Guide , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Replication/drug effects
3.
Nat Commun ; 13(1): 1937, 2022 04 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
4.
Nat Commun ; 13(1): 1722, 2022 03 31.
Article in English | MEDLINE | ID: covidwho-1773975

ABSTRACT

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes.


Subject(s)
Zika Virus Infection , Zika Virus , Algorithms , Humans , Nucleic Acid Conformation , RNA/chemistry , RNA/genetics , RNA, Guide , Sequence Analysis, RNA/methods , Zika Virus/genetics
5.
EBioMedicine ; 77: 103926, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1739672

ABSTRACT

BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).


Subject(s)
Brevibacillus , COVID-19 , Brevibacillus/genetics , COVID-19/diagnosis , Humans , RNA, Guide , SARS-CoV-2/genetics
6.
Nucleic Acids Res ; 50(6): 3475-3489, 2022 04 08.
Article in English | MEDLINE | ID: covidwho-1730702

ABSTRACT

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.


Subject(s)
COVID-19 , Nanopores , RNA, Guide/chemistry , COVID-19/genetics , Genome, Viral/genetics , Humans , RNA Caps , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics
7.
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
8.
Nucleic Acids Res ; 50(8): e47, 2022 05 06.
Article in English | MEDLINE | ID: covidwho-1684755

ABSTRACT

Gene-editing technologies, including the widespread usage of CRISPR endonucleases, have the potential for clinical treatments of various human diseases. Due to the rapid mutations of SARS-CoV-2, specific and effective prevention and treatment by CRISPR toolkits for coronavirus disease 2019 (COVID-19) are urgently needed to control the current pandemic spread. Here, we designed Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) as a therapeutic to combat SARS-CoV-2 infection. We provided a proof of principle demonstration that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses. We report that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice. Moreover, bioinformatics screening of gRNAs along RNA sequences reveals that a group of five gRNAs (hCoV-gRNAs) could potentially target 99.98% of human coronaviruses. TEAR-CoV also exerted specific targeting and cleavage of common human coronaviruses. The fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antiviral Agents , COVID-19/therapy , Humans , Mice , Pandemics/prevention & control , RNA Editing/genetics , RNA, Guide/genetics , SARS-CoV-2/genetics
9.
Curr Top Med Chem ; 21(32): 2851-2855, 2021.
Article in English | MEDLINE | ID: covidwho-1628408

ABSTRACT

In this era, RNA molecules have provided a unique opportunity to researchers all over the world for expanding their range of targets in the development of drugs. Due to the unique pharmacological as well as physicochemical characteristics of different RNA molecules such as aptamers, small interfering RNAs (siRNA), antisense oligonucleotides (ASO) and guide RNAs (gRNA), they have emerged recently as a new class of drugs. They are used for selective action on proteins and genes that were not possible to target by conventional drug molecules. These RNA molecules like guide RNAs are also components of novel gene editing mechanisms which can modify the genome nearly in all cells. Vaccines based on RNA molecules have also provided a promising alternative to conventional live attenuated vaccines. RNA based vaccines have high potency, can be rapidly developed, and have potential for manufacturing at a cheaper rate and safe administration. However, the application of these RNAs has been restricted by the high instability and inefficient in vivo delivery. Technological advancement needs to overcome these issues so that RNA based drugs targeting several diseases can be developed. This article emphasizes the potential of RNA based drugs and the major barriers associated with the development of RNA therapeutics. Additionally, the role of RNA based vaccines and their challenges in advancing this promising vaccine platform for the prevention of infectious diseases have been discussed.


Subject(s)
Aptamers, Nucleotide , Oligonucleotides, Antisense , Pharmaceutical Preparations/classification , RNA, Guide , RNA, Small Interfering , Animals , Humans
10.
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
11.
Chem Pharm Bull (Tokyo) ; 69(12): 1141-1159, 2021.
Article in English | MEDLINE | ID: covidwho-1546823

ABSTRACT

Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.


Subject(s)
Drug Carriers/chemistry , Lipids/chemistry , Liposomes/chemistry , Nanoparticles/chemistry , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Cations/chemistry , Hydrogen-Ion Concentration , RNA, Guide/chemistry , RNA, Guide/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , SARS-CoV-2/isolation & purification , /metabolism
12.
Biomolecules ; 11(11)2021 11 17.
Article in English | MEDLINE | ID: covidwho-1523862

ABSTRACT

Metal-organic frameworks (MOFs) have been widely used as porous nanomaterials for different applications ranging from industrial to biomedicals. An unpredictable one-pot method is introduced to synthesize NH2-MIL-53 assisted by high-gravity in a greener media for the first time. Then, porphyrins were deployed to adorn the surface of MOF to increase the sensitivity of the prepared nanocomposite to the genetic materials and in-situ cellular protein structures. The hydrogen bond formation between genetic domains and the porphyrin' nitrogen as well as the surface hydroxyl groups is equally probable and could be considered a milestone in chemical physics and physical chemistry for biomedical applications. In this context, the role of incorporating different forms of porphyrins, their relationship with the final surface morphology, and their drug/gene loading efficiency were investigated to provide a predictable pattern in regard to the previous works. The conceptual phenomenon was optimized to increase the interactions between the biomolecules and the substrate by reaching the limit of detection to 10 pM for the Anti-cas9 protein, 20 pM for the single-stranded DNA (ssDNA), below 10 pM for the single guide RNA (sgRNA) and also around 10 nM for recombinant SARS-CoV-2 spike antigen. Also, the MTT assay showed acceptable relative cell viability of more than 85% in most cases, even by increasing the dose of the prepared nanostructures.


Subject(s)
COVID-19/diagnosis , Metal-Organic Frameworks/chemistry , Porphyrins/chemistry , Animals , COVID-19 Testing , CRISPR-Cas Systems , DNA, Single-Stranded , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Hydrogen Bonding , Limit of Detection , Nanocomposites , Nanostructures , Nitrogen/chemistry , PC12 Cells , Porosity , RNA, Guide , RNA, Viral/metabolism , Rats , SARS-CoV-2 , Sensitivity and Specificity , Surface Properties
13.
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
14.
Bioessays ; 43(4): e2000315, 2021 04.
Article in English | MEDLINE | ID: covidwho-1384113

ABSTRACT

The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA- and RNA-targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral nucleic acids in "Point of Care" sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS-CoV-2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR-based viral detectors. Finally, we discuss future prospects to improve both applications. Also see the video abstract here: https://www.youtube.com/watch?v=C0z1dLpJWl4.


Subject(s)
Biosensing Techniques/methods , CRISPR-Cas Systems , Virus Diseases/diagnosis , Virus Diseases/therapy , Viruses/genetics , COVID-19/diagnosis , COVID-19/genetics , COVID-19/therapy , Gene Knock-In Techniques , Genome, Viral , Humans , RNA, Guide/genetics
15.
Biosensors (Basel) ; 11(9)2021 Aug 28.
Article in English | MEDLINE | ID: covidwho-1374295

ABSTRACT

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.


Subject(s)
Bacterial Proteins/metabolism , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/metabolism , Middle East Respiratory Syndrome Coronavirus/isolation & purification , SARS Virus/isolation & purification , SARS-CoV-2/isolation & purification , Virus Diseases/diagnosis , CRISPR-Cas Systems , Fluorescent Dyes/chemistry , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Nose/virology , Point-of-Care Testing , RNA, Guide/chemistry , RNA, Guide/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS Virus/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Sputum/virology
16.
Front Immunol ; 12: 655122, 2021.
Article in English | MEDLINE | ID: covidwho-1365539

ABSTRACT

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Subject(s)
Gene Editing/methods , Interleukin-2 Receptor alpha Subunit/genetics , Receptors, Interleukin-6/genetics , T-Lymphocytes, Regulatory/metabolism , Blood Buffy Coat/cytology , CRISPR-Cas Systems/genetics , Forkhead Transcription Factors/metabolism , Gene Knockdown Techniques , HEK293 Cells , Healthy Volunteers , Humans , Immunotherapy, Adoptive/methods , Primary Cell Culture , RNA, Guide/genetics , Time Factors
17.
Mol Cell ; 81(17): 3650-3658.e5, 2021 09 02.
Article in English | MEDLINE | ID: covidwho-1356368

ABSTRACT

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.


Subject(s)
Epitopes/genetics , Gene Editing/methods , Peptide Library , RNA, Guide/genetics , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/immunology , Epitopes/immunology , Humans , Mutagenesis/genetics , Protein Binding/genetics , Protein Binding/immunology , RNA, Guide/immunology
18.
Cell ; 184(16): 4203-4219.e32, 2021 08 05.
Article in English | MEDLINE | ID: covidwho-1275187

ABSTRACT

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Viral/immunology , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Cytokines/metabolism , Female , Haplorhini , Humans , Lung/pathology , Lung/virology , Male , Mice , Mice, Inbred BALB C , Protein Domains , RNA, Guide/metabolism , Receptors, IgG/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Viral Load , Virus Replication
19.
Cell Rep Med ; 2(6): 100319, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1244849

ABSTRACT

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , RNA, Viral/metabolism , COVID-19/virology , Colorimetry , Humans , Molecular Diagnostic Techniques , Nasopharynx/virology , Nucleic Acid Amplification Techniques , RNA, Guide/metabolism , RNA, Viral/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism
20.
Science ; 372(6545): 941-948, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1205995

ABSTRACT

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.


Subject(s)
CRISPR-Associated Protein 9/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , RNA, Guide/genetics , RNA, Viral/analysis , RNA/analysis , RNA/genetics , SARS-CoV-2/genetics , Base Sequence , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing , CRISPR-Cas Systems , Campylobacter jejuni , Humans , Nucleic Acid Hybridization , RNA, Bacterial/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Spike Glycoprotein, Coronavirus/genetics
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