RESUMO
ACE2 is the indispensable entry receptor for SARS-CoV and SARS-CoV-2. Because of the COVID-19 pandemic, it has become one of the most therapeutically targeted human molecules in biomedicine. ACE2 serves two fundamental physiological roles: as an enzyme, it alters peptide cascade balance; as a chaperone, it controls intestinal amino acid uptake. ACE2's tissue distribution, affected by co-morbidities and sex, explains the broad tropism of coronaviruses and the clinical manifestations of SARS and COVID-19. ACE2-based therapeutics provide a universal strategy to prevent and treat SARS-CoV-2 infections, applicable to all SARS-CoV-2 variants and other emerging zoonotic coronaviruses exploiting ACE2 as their cellular receptor.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2 , Peptidil Dipeptidase A/metabolismo , PandemiasRESUMO
New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.
Assuntos
Receptores Mitogênicos/metabolismo , SARS-CoV-2/metabolismo , Animais , Sítios de Ligação/fisiologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Glicosilação , Células HEK293 , Humanos , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica/fisiologia , Células Vero , Internalização do VírusRESUMO
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Silenciamento de Genes/métodos , Leptotrichia/enzimologia , RNA/genética , RNA/metabolismo , Biocatálise , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Escherichia coli/genética , Genes Reporter/genética , Células HEK293 , Humanos , Leptotrichia/genética , Células Vegetais/metabolismo , RNA/análise , Interferência de RNA , Estresse Fisiológico , Especificidade por SubstratoRESUMO
MicroRNAs (miRNAs) are short noncoding RNAs that mediate the repression of target transcripts in plants and animals. Although miRNAs are required throughout plant development, relatively little is known regarding their embryonic functions. To systematically characterize embryonic miRNAs in Arabidopsis (Arabidopsis thaliana), we developed or applied high-throughput sequencing-based methods to profile hundreds of miRNAs and associated targets throughout embryogenesis. We discovered dozens of miRNAs that dynamically cleave and repress target transcripts, including 30 that encode transcription factors. Transcriptome analyses indicated that these miRNA:target interactions have profound effects on embryonic gene expression programs. Moreover, we demonstrated that the miRNA-mediated repression of six transcription factors are individually required for proper division patterns of various embryonic cell lineages. These data indicate that the miRNA-directed repression of multiple transcription factors is critically important for the establishment of the plant body plan, and they provide a foundation to further investigate how miRNAs contribute to these initial cellular differentiation events.
Assuntos
Arabidopsis/embriologia , Arabidopsis/metabolismo , MicroRNAs/metabolismo , Sementes/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Morfogênese/genética , Morfogênese/fisiologia , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas , Sementes/genética , Fatores de Transcrição/genética , Transcriptoma/fisiologiaRESUMO
Diverse RNA 5' ends are generated through both transcriptional and post-transcriptional processes. These important modes of gene regulation often vary across cell types and can contribute to the diversification of transcriptomes and thus cellular differentiation. Therefore, the identification of primary and processed 5' ends of RNAs is important for their functional characterization. Methods have been developed to profile either RNA 5' ends from primary transcripts or the products of RNA degradation genome-wide. However, these approaches either require high amounts of starting RNA or are performed in the absence of paired gene-body mRNA-seq data. This limits current efforts in RNA 5' end annotation to whole tissues and can prevent accurate RNA 5' end classification due to biases in the data sets. To enable the accurate identification and precise classification of RNA 5' ends from standard and low-input RNA, we developed a next-generation sequencing-based method called nanoPARE and associated software. By integrating RNA 5' end information from nanoPARE with gene-body mRNA-seq data from the same RNA sample, our method enables the identification of transcription start sites at single-nucleotide resolution from single-cell levels of total RNA, as well as small RNA-mediated cleavage events from at least 10,000-fold less total RNA compared to conventional approaches. NanoPARE can therefore be used to accurately profile transcription start sites, noncapped RNA 5' ends, and small RNA targeting events from individual tissue types. As a proof-of-principle, we utilized nanoPARE to improve Arabidopsis thaliana RNA 5' end annotations and quantify microRNA-mediated cleavage events across five different flower tissues.
Assuntos
Regiões 5' não Traduzidas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanotecnologia , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , MicroRNAs/genética , Clivagem do RNA , Interferência de RNA , Sítio de Iniciação de TranscriçãoRESUMO
Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.
Assuntos
Apolipoproteínas E , Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Receptores de LDL , Internalização do Vírus , Animais , Humanos , Camundongos , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/virologia , Febre Hemorrágica da Crimeia/metabolismo , Camundongos Knockout , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virais/metabolismo , Carrapatos/virologia , Carrapatos/metabolismoRESUMO
Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Doença do Vírus de Marburg , Marburgvirus , Proteínas de Transporte Vesicular , Animais , Humanos , Ebolavirus/metabolismo , Lisossomos , Doença do Vírus de Marburg/genética , Doença do Vírus de Marburg/metabolismo , Marburgvirus/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.
RESUMO
The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here, we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.
Assuntos
Enzima de Conversão de Angiotensina 2 , Tratamento Farmacológico da COVID-19 , Humanos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, leads to respiratory symptoms that can be fatal. However, neurological symptoms have also been observed in some patients. The cause of these complications is currently unknown. Here, we use human-pluripotent-stem-cell-derived brain organoids to examine SARS-CoV-2 neurotropism. We find expression of viral receptor ACE2 in mature choroid plexus cells expressing abundant lipoproteins, but not in neurons or other cell types. We challenge organoids with SARS-CoV-2 spike pseudovirus and live virus to demonstrate viral tropism for choroid plexus epithelial cells but little to no infection of neurons or glia. We find that infected cells are apolipoprotein- and ACE2-expressing cells of the choroid plexus epithelial barrier. Finally, we show that infection with SARS-CoV-2 damages the choroid plexus epithelium, leading to leakage across this important barrier that normally prevents entry of pathogens, immune cells, and cytokines into cerebrospinal fluid and the brain.
Assuntos
Barreira Hematoencefálica/virologia , Plexo Corióideo/virologia , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Modelos Biológicos , Organoides/virologia , Células Vero , Tropismo Viral , Internalização do VírusRESUMO
Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (â¼15 min), quantitative, and portable, and can process crude soybean extracts as input material for minimal nucleic acid sample preparation. This field-ready SHERLOCK platform with color-based lateral flow readout can be applied for detection and quantitation of genes in a range of agricultural applications.
Assuntos
Sistemas CRISPR-Cas , DNA de Plantas/análise , Genes de Plantas , Testes Genéticos/métodos , Glycine max/genética , EndodesoxirribonucleasesRESUMO
Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.
Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Ácidos Nucleicos/análise , Primers do DNA , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Humanos , Leptotrichia/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação , Ribonucleases/metabolismo , Fluxo de Trabalho , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urinaRESUMO
Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive ß-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.
Assuntos
Adenosina Desaminase/genética , Citidina/genética , Citosina Desaminase/genética , Engenharia de Proteínas/métodos , Edição de RNA , Proteínas de Ligação a RNA/genética , Uridina/genética , Adenosina/genética , Adenosina Desaminase/química , Citosina Desaminase/química , Células HEK293 , Humanos , Inosina/genética , Domínios Proteicos , Proteínas de Ligação a RNA/química , beta Catenina/química , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.
Assuntos
Proteínas de Bactérias/química , Sistemas CRISPR-Cas , Endonucleases/química , Prevotella/enzimologia , RNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Endonucleases/genética , Endonucleases/metabolismo , Estabilidade Enzimática , Ligação Proteica , Domínios Proteicos , RNA/química , Especificidade por SubstratoRESUMO
Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , DNA/análise , Endonucleases/química , Ensaios Enzimáticos , RNA/análise , Vírus da Dengue/isolamento & purificação , Humanos , RNA Viral/análise , Sensibilidade e Especificidade , Zika virus/isolamento & purificaçãoRESUMO
Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015-2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Endonucleases/química , Ensaios Enzimáticos , RNA Viral/análise , Infecção por Zika virus/diagnóstico , Zika virus/isolamento & purificação , Adaptação Fisiológica/genética , Vírus da Dengue/genética , Humanos , Microcefalia/diagnóstico , Microcefalia/virologia , Polimorfismo de Nucleotídeo Único , Zika virus/genéticaRESUMO
Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.