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1.
GM Crops Food ; 12(1): 125-144, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-33079628

RESUMO

Plants are susceptible to phytopathogens, including bacteria, fungi, and viruses, which cause colossal financial shortfalls (pre- and post-harvest) and threaten global food safety. To combat with these phytopathogens, plant possesses two-layer of defense in the form of PAMP-triggered immunity (PTI), or Effectors-triggered immunity (ETI). The understanding of plant-molecular interactions and revolution of high-throughput molecular techniques have opened the door for innovations in developing pathogen-resistant plants. In this context, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has transformed genome editing (GE) technology and being harnessed for altering the traits. Here we have summarized the complexities of plant immune system and the use of CRISPR-Cas9 to edit the various components of plant immune system to acquire long-lasting resistance in plants against phytopathogens. This review also sheds the light on the limitations of CRISPR-Cas9 system, regulation of CRISPR-Cas9 edited crops and future prospective of this technology.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Produtos Agrícolas/genética , Edição de Genes , Doenças das Plantas/genética
2.
GM Crops Food ; 12(1): 57-70, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32877304

RESUMO

Hybrid seeds of several important crops with supreme qualities including yield, biotic and abiotic stress tolerance have been cultivated for decades. Thus far, a major challenge with hybrid seeds is that they do not have the ability to produce plants with the same qualities over subsequent generations. Apomixis, an asexual mode of reproduction by avoiding meiosis, exists naturally in flowering plants, and ultimately leads to seed production. Apomixis has the potential to preserve hybrid vigor for multiple generations in economically important plant genotypes. The evolution and genetics of asexual seed production are unclear, and much more effort will be required to determine the genetic architecture of this phenomenon. To fix hybrid vigor, synthetic apomixis has been suggested. The development of MiMe (mitosis instead of meiosis) genotypes has been utilized for clonal gamete production. However, the identification and parental origin of genes responsible for synthetic apomixis are little known and need further clarification. Genome modifications utilizing genome editing technologies (GETs), such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (cas), a reverse genetics tool, have paved the way toward the utilization of emerging technologies in plant molecular biology. Over the last decade, several genes in important crops have been successfully edited. The vast availability of GETs has made functional genomics studies easy to conduct in crops important for food security. Disruption in the expression of genes specific to egg cell MATRILINEAL (MTL) through the CRISPR/Cas genome editing system promotes the induction of haploid seed, whereas triple knockout of the Baby Boom (BBM) genes BBM1, BBM2, and BBM3 cause embryo arrest and abortion, which can be fully rescued by male-transmitted BBM1. The establishment of synthetic apomixis by engineering the MiMe genotype by genome editing of BBM1 expression or disruption of MTL leads to clonal seed production and heritability for multiple generations. In the present review, we discuss current developments related to the use of CRISPR/Cas technology in plants and the possibility of promoting apomixis in crops to preserve hybrid vigor. In addition, genetics, evolution, epigenetic modifications, and strategies for MiMe genotype development are discussed in detail.


Assuntos
Apomixia , Sistemas CRISPR-Cas , Produtos Agrícolas/genética , Edição de Genes , Vigor Híbrido , Sementes
4.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
5.
Nat Commun ; 11(1): 4906, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999292

RESUMO

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Transativadores/metabolismo , Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , DNA de Cadeia Simples , Pandemias , Pneumonia Viral , RNA Guia/genética , RNA Viral/genética
6.
Nat Commun ; 11(1): 5060, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033246

RESUMO

Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.


Assuntos
Sistemas CRISPR-Cas/genética , Neoplasias/genética , Fusão Oncogênica/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Doxorrubicina/uso terapêutico , Proteínas de Fusão bcr-abl/genética , Deleção de Genes , Loci Gênicos , Instabilidade Genômica , Células HEK293 , Humanos , Íntrons/genética , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas de Fusão Oncogênica/genética , RNA Guia/metabolismo , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS Comput Biol ; 16(9): e1008194, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936799

RESUMO

CRISPR screens are a powerful technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By targeting non-coding sequences for perturbation, CRISPR screens have the potential to systematically discover novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from CRISPR screens. RELICS specifically addresses many of the challenges of non-coding CRISPR screens such as the unknown locations of functional sequences, overdispersion in the observed single guide RNA counts, and the need to combine information across multiple pools in an experiment. RELICS outperforms existing methods with higher precision, higher recall, and finer-resolution predictions on simulated datasets. We apply RELICS to published CRISPR interference and CRISPR activation screens to predict and experimentally validate novel regulatory sequences that are missed by other analysis methods. In summary, RELICS is a powerful new analysis method for CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.


Assuntos
Sistemas CRISPR-Cas/genética , Genômica/métodos , Análise de Sequência de DNA/métodos , Software , Teorema de Bayes , Humanos , Células Jurkat , RNA Guia/genética
8.
PLoS Pathog ; 16(9): e1008834, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956422

RESUMO

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação Viral da Expressão Gênica , Inativação Gênica , HIV-1/fisiologia , Proteínas Repressoras , Fatores de Transcrição , Transcrição Genética , Latência Viral , Linfócitos T CD4-Positivos/virologia , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Humanos , Células Jurkat , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
9.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
10.
Nat Commun ; 11(1): 4820, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973160

RESUMO

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.


Assuntos
Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/metabolismo , Animais , Sistemas CRISPR-Cas , Quimiocinas/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tirosina-tRNA Ligase/química
11.
Tumour Biol ; 42(9): 1010428320962588, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32996421

RESUMO

A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H-mutated (GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.


Assuntos
Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Mutação , Teratoma/patologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Cromograninas/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos SCID , Teratoma/genética
12.
Nat Commun ; 11(1): 4464, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900994

RESUMO

The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Bioensaio , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
13.
Nat Commun ; 11(1): 4871, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978399

RESUMO

Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.


Assuntos
Adenina/química , Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , RNA/metabolismo , Proteína 9 Associada à CRISPR , Citosina , DNA , Exoma , Genoma , Células HEK293 , Humanos , Edição de RNA
15.
Nature ; 585(7826): 603-608, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939090

RESUMO

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.


Assuntos
Éteres/metabolismo , Ferroptose , Peroxissomos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem Celular , Éteres/química , Feminino , Edição de Genes , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Peroxidação de Lipídeos , Masculino , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Peroxissomos/genética
16.
Nat Commun ; 11(1): 4711, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948757

RESUMO

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Betacoronavirus/genética , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Genes Virais , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Proteínas Virais/análise , Proteínas Virais/genética
17.
Biosens Bioelectron ; 169: 112642, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979593

RESUMO

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/genética , Técnicas Biossensoriais/instrumentação , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/virologia , Desenho de Equipamento , Fluorescência , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Smartphone
18.
Mol Cell ; 79(5): 703-704, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32888434

RESUMO

Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas , Citosina , DNA de Cadeia Simples , Desaminação
19.
PLoS Negl Trop Dis ; 14(8): e0008627, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866158

RESUMO

The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.


Assuntos
Brugia Malayi/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transfecção/métodos , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA de Helmintos/genética , Feminino , Edição de Genes , Genoma , Larva/genética , Luciferases , Microfilárias/genética
20.
Nat Commun ; 11(1): 4581, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917893

RESUMO

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Oncogenes/genética , Fatores de Transcrição/genética
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