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1.
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
2.
PLoS Pathog ; 18(3): e1010377, 2022 03.
Article in English | MEDLINE | ID: covidwho-1714786

ABSTRACT

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.


Subject(s)
COVID-19 , Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Cell Line , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
3.
FASEB J ; 36(3): e22234, 2022 03.
Article in English | MEDLINE | ID: covidwho-1702985

ABSTRACT

The transmembrane protease angiotensin converting enzyme 2 (ACE2) is a protective regulator within the renin angiotensin system and additionally represents the cellular receptor for SARS-CoV. The release of soluble ACE2 (sACE2) from the cell surface is hence believed to be a crucial part of its (patho)physiological functions, as both, ACE2 protease activity and SARS-CoV binding ability, are transferred from the cell membrane to body fluids. Yet, the molecular sources of sACE2 are still not completely investigated. In this study, we show different sources and prerequisites for the release of sACE2 from the cell membrane. By using inhibitors as well as CRISPR/Cas9-derived cells, we demonstrated that, in addition to the metalloprotease ADAM17, also ADAM10 is an important novel shedding protease of ACE2. Moreover, we observed that ACE2 can also be released in extracellular vesicles. The degree of either ADAM10- or ADAM17-mediated ACE2 shedding is dependent on stimulatory conditions and on the expression level of the pro-inflammatory ADAM17 regulator iRhom2. Finally, by using structural analysis and in vitro verification, we determined for the first time that the susceptibility to ADAM10- and ADAM17-mediated shedding is mediated by the collectrin-like part of ACE2. Overall, our findings give novel insights into sACE2 release by several independent molecular mechanisms.


Subject(s)
ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Extracellular Vesicles/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , SARS Virus/metabolism , ADAM10 Protein/genetics , ADAM17 Protein/genetics , Amyloid Precursor Protein Secretases/genetics , Angiotensin-Converting Enzyme 2/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Extracellular Vesicles/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , SARS Virus/genetics , SARS-CoV-2
4.
Biosens Bioelectron ; 202: 113978, 2022 Apr 15.
Article in English | MEDLINE | ID: covidwho-1661800

ABSTRACT

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


Subject(s)
Biosensing Techniques , COVID-19 , Quantum Dots , Chromatography, Affinity , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
5.
Biosens Bioelectron ; 201: 113960, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1633190

ABSTRACT

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


Subject(s)
Biosensing Techniques , COVID-19 , COVID-19 Testing , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques , Pandemics , RNA, Viral/genetics , SARS-CoV-2
6.
Bioprocess Biosyst Eng ; 45(3): 503-514, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1627214

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had severe consequences for health and the global economy. To control the transmission, there is an urgent demand for early diagnosis and treatment in the general population. In the present study, an automatic system for SARS-CoV-2 diagnosis is designed and built to deliver high specification, high sensitivity, and high throughput with minimal workforce involvement. The system, set up with cross-priming amplification (CPA) rather than conventional reverse transcription-polymerase chain reaction (RT-PCR), was evaluated using more than 1000 real-world samples for direct comparison. This fully automated robotic system performed SARS-CoV-2 nucleic acid-based diagnosis with 192 samples in under 180 min at 100 copies per reaction in a "specimen in data out" manner. This throughput translates to a daily screening capacity of 800-1000 in an assembly-line manner with limited workforce involvement. The sensitivity of this device could be further improved using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based assay, which opens the door to mixed samples, potentially include SARS-CoV-2 variants screening in extensively scaled testing for fighting COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2 , Algorithms , Biomedical Engineering/instrumentation , Biomedical Engineering/methods , Biomedical Engineering/statistics & numerical data , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/statistics & numerical data , Clustered Regularly Interspaced Short Palindromic Repeats , Equipment Design , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , High-Throughput Screening Assays/statistics & numerical data , Humans , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/statistics & numerical data , Pandemics , Robotics/instrumentation , Robotics/methods , Robotics/statistics & numerical data , SARS-CoV-2/genetics , Sensitivity and Specificity , Systems Analysis
7.
J Mol Biol ; 434(5): 167403, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1611867

ABSTRACT

COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.


Subject(s)
COVID-19/virology , Host Microbial Interactions , Host-Derived Cellular Factors/metabolism , Pandemics , SARS-CoV-2/physiology , COVID-19/epidemiology , Clustered Regularly Interspaced Short Palindromic Repeats , Host-Derived Cellular Factors/genetics , Humans , SARS-CoV-2/genetics
9.
J Clin Lab Anal ; 36(1): e24178, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1589069

ABSTRACT

Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, it has been clear that effective methods for the diagnosis of Corona Virus Disease 2019 (COVID-19) are the key tools to control its epidemic. The current gold standard for diagnosing COVID-19 is the real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), which is a sensitive and specific method to detect SARS-CoV-2. Other RNA-based methods include RNA sequencing (RNA-seq), droplet digital reverse transcription-polymerase chain reaction (ddRT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR). The serological testing of antibodies (IgM and IgG), nanoparticle-based lateral-flow assay, and enzyme-linked immunosorbent assay (ELISA) can be used to enhance the detection sensitivity and accuracy. Because antibodies are usually detected a week after the onset of symptoms, these tests are used to assess the overall infection rate in the community. Sine the fact that healthcare varies from country to country across the world, different types of diagnosing COVID-19 imaging technologies including chest computed tomography (CT), chest radiography, and lung ultrasound are used in different degrees. Besides, the pooling test is an important public health tool to reduce cost and increase testing capacity in low-risk area, while artificial intelligence (AI) may aid to increase the diagnostic efficiency of imaging-based methods. Finally, depending on the type of samples and stages of the disease, a combination of information on patient demographics and histories, clinical symptoms, results of molecular and serological diagnostic tests, and imaging information is highly recommended to achieve adequate diagnosis of patients with COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Antibodies, Viral/blood , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Enzyme-Linked Immunosorbent Assay/methods , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , RNA-Seq/methods , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2 , Sensitivity and Specificity
11.
Methods Mol Biol ; 2410: 209-228, 2022.
Article in English | MEDLINE | ID: covidwho-1576030

ABSTRACT

The COVID-19 pandemic brought to the fore the urgent need for vaccine design and delivery platforms that can be rapidly deployed for manufacture and distribution. Though the mRNA and adenoviral vector platforms have been enormously successful to control SARS-CoV-2 viral infections, it is unclear if this could be replicated against more complex pathogens or the emerging variants. Recently, we described a "universal" platform that can incorporate multiple vaccine targets into the same nanoparticle scaffold by CRISPR engineering of bacteriophage T4. A T4-COVID vaccine designed with this technology elicited broad immunogenicity and complete protection against virus challenge in a mouse model. Here, we describe the detailed methodology to generate recombinant bacteriophage T4 backbones using CRISPR that can also be broadly applicable to other bacteriophages that abundantly pervade the Earth.


Subject(s)
Bacteriophage T4 , COVID-19 Vaccines , COVID-19 , Clustered Regularly Interspaced Short Palindromic Repeats , Animals , Bacteriophage T4/genetics , COVID-19/prevention & control , Humans , Mice , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
12.
Horm Mol Biol Clin Investig ; 43(1): 105-112, 2021 Dec 08.
Article in English | MEDLINE | ID: covidwho-1559691

ABSTRACT

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a novel molecular tool. In recent days, it has been highlighted a lot, as the Nobel prize was awarded for this sector in 2020, and also for its recent use in Covid-19 related diagnostics. Otherwise, it is an eminent gene-editing technique applied in diverse medical zones of therapeutics in genetic diseases, hematological diseases, infectious diseases, etc., research related to molecular biology, cancer, hereditary diseases, immune and inflammatory diseases, etc., diagnostics related to infectious diseases like viral hemorrhagic fevers, Covid-19, etc. In this review, its discovery, working mechanisms, challenges while handling the technique, recent advancements, applications, alternatives have been discussed. It is a cheaper, faster technique revolutionizing the medicinal field right now. However, their off-target effects and difficulties in delivery into the desired cells make CRISPR, not easily utilizable. We conclude that further robust research in this field may promise many interesting, useful results.


Subject(s)
COVID-19 , Clustered Regularly Interspaced Short Palindromic Repeats , COVID-19/diagnosis , CRISPR-Cas Systems , Genetic Therapy/methods , Humans , Molecular Biology , SARS-CoV-2/genetics
13.
J Immunol ; 208(1): 74-84, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1534334

ABSTRACT

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , Interferon Type I/metabolism , Lung/immunology , Neoplasm Proteins/metabolism , ORAI1 Protein/metabolism , Respiratory Mucosa/metabolism , SARS-CoV-2/physiology , Stromal Interaction Molecule 1/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , Calcium Signaling , Clustered Regularly Interspaced Short Palindromic Repeats , Disease Resistance , Disease Susceptibility , Gene Expression Profiling , HEK293 Cells , Humans , Lung/virology , MEF2 Transcription Factors/genetics , Neoplasm Proteins/genetics , ORAI1 Protein/genetics , Stromal Interaction Molecule 1/genetics , Transcription Factor AP-1/genetics
14.
Elife ; 102021 04 27.
Article in English | MEDLINE | ID: covidwho-1513055

ABSTRACT

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.


Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Dendritic Cells/immunology , Gene Editing , Genomics , Immunity, Innate/genetics , Bacteroides thetaiotaomicron/immunology , CRISPR-Associated Protein 9/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Gene Expression Regulation , Humans , Immunity, Innate/drug effects , Lipopolysaccharides/pharmacology , Phenotype , Signal Transduction , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism
15.
Science ; 372(6545): 914-915, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1501517
16.
Viruses ; 13(11)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1488755

ABSTRACT

Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.


Subject(s)
CRISPR-Cas Systems , Communicable Diseases, Emerging/virology , Coronavirus Infections/virology , Coronavirus/genetics , Gene Editing , Zika Virus Infection/virology , Zika Virus/genetics , COVID-19/diagnosis , COVID-19/virology , Clustered Regularly Interspaced Short Palindromic Repeats , Communicable Diseases, Emerging/diagnosis , Coronavirus/physiology , Coronavirus Infections/diagnosis , Host-Pathogen Interactions , Humans , SARS-CoV-2/genetics , Zika Virus/physiology , Zika Virus Infection/diagnosis
18.
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
20.
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