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1.
Nat Commun ; 13(1): 2766, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35589813

RESUMO

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Humanos , Lipossomos , Nanopartículas , SARS-CoV-2/genética
2.
Cell ; 181(4): 865-876.e12, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32353252

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Sistemas CRISPR-Cas , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Células A549 , Antibioticoprofilaxia/métodos , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , COVID-19 , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Simulação por Computador , Sequência Conservada , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Coronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/tratamento farmacológico , Proteínas do Nucleocapsídeo de Coronavírus , RNA-Polimerase RNA-Dependente de Coronavírus , Células Epiteliais/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Filogenia , Pneumonia Viral/tratamento farmacológico , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Proteínas não Estruturais Virais/genética
3.
Science ; 365(6459): 1301-1305, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31488703

RESUMO

We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Hibridização in Situ Fluorescente/métodos , Linhagem Celular Tumoral , DNA/análise , Quebras de DNA de Cadeia Dupla , Vetores Genéticos , Células HEK293 , Humanos , Microscopia de Fluorescência , Imagem Molecular , RNA/análise , RNA Guia de Cinetoplastídeos/genética , Linfócitos T
4.
Nat Commun ; 10(1): 194, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643127

RESUMO

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.


Assuntos
Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Redes Reguladoras de Genes/genética , Técnicas de Cultura de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Eucarióticas , Vetores Genéticos/genética , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Microscopia Intravital/métodos , Lentivirus/genética , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodos , Transdução Genética/métodos , Transfecção/métodos
5.
Mol Cell ; 72(3): 402-403, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388408

RESUMO

In a recent issue of Nature, Halperin et al. (2018) develop a new technology to continuously diversify specific genomic loci by combining CRISPR-Cas9 with error-prone DNA polymerases.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Polimerase Dirigida por DNA , Genômica , Nucleotídeos
6.
Nat Commun ; 8(1): 2212, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29263378

RESUMO

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.


Assuntos
Sistemas CRISPR-Cas , Engenharia Celular/métodos , Receptores Acoplados a Proteínas G , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HEK293 , Humanos , Ligantes , Modelos Teóricos
7.
RNA ; 22(6): 920-33, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27103533

RESUMO

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators.


Assuntos
Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , RNA Bacteriano/genética , Transcrição Gênica , Escherichia coli/genética , Simulação de Dinâmica Molecular , Mutação
8.
Nucleic Acids Res ; 44(2): e12, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26350218

RESUMO

Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure-function relationship of two RNA mechanisms that regulate translation in Escherichia coli. We find that nucleotides that participate in RNA-RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA-RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/química , RNA Ribossômico 5S/química , Ribonuclease P/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , Ribonuclease P/genética , Ribossomos/química , Ribossomos/genética , Riboswitch , Análise de Sequência de RNA , Relação Estrutura-Atividade
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