Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 64
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nucleic Acids Res ; 52(10): 6079-6091, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38661215

RESUMO

CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.


Assuntos
Sistemas CRISPR-Cas , Klebsiella pneumoniae , RNA Guia de Sistemas CRISPR-Cas , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma Bacteriano/genética , Edição de Genes/métodos , Humanos
2.
Methods Mol Biol ; 2793: 85-100, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38526725

RESUMO

Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.


Assuntos
Bacteriófago T7 , Bacteriófagos , Bacteriófago T7/genética , Lipopolissacarídeos/metabolismo , Bacteriófagos/genética , Ligação Proteica , Proteínas de Transporte/metabolismo , Especificidade de Hospedeiro
3.
Microbiol Spectr ; 11(6): e0169723, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-37888989

RESUMO

IMPORTANCE: We have identified a novel phage-encoded inhibitor of the major cytoskeletal protein in bacterial division, FtsZ. The inhibition is shown to confer T5 bacteriophage with a growth advantage in dividing hosts. Our studies demonstrate a strategy in bacteriophages to maximize their progeny number by inhibiting escape of one of the daughter cells of an infected bacterium. They further emphasize that FtsZ is a natural target for bacterial growth inhibition.


Assuntos
Bacteriófagos , Divisão Celular , Bacteriófagos/fisiologia , Bactérias , Proteínas do Citoesqueleto , Proteínas de Bactérias/genética
4.
RNA Biol ; 20(1): 830-835, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-37846029

RESUMO

Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50-201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text].


Assuntos
Bacteriófagos , Bacteriófagos/genética , Escherichia coli/genética , Sistemas CRISPR-Cas , Mutação , Tecnologia
5.
Nucleic Acids Res ; 51(14): 7552-7562, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37326009

RESUMO

Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.


Assuntos
Proteínas Associadas a CRISPR , Proteínas de Escherichia coli , Escherichia coli , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética
6.
Nat Microbiol ; 8(3): 410-423, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36759752

RESUMO

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.


Assuntos
Bacteriófagos , Genes Bacterianos , Antibacterianos/farmacologia , Metagenômica , Bacteriófagos/genética , Bactérias/genética
7.
Mol Cell ; 82(23): 4403-4404, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36459982

RESUMO

Wu et al.1 characterize Cas12m, a compact Cas protein that silences transcription without cleaving DNA and is a prototype protein of the novel CRISPR-Cas subtype V-M.


Assuntos
Sistemas CRISPR-Cas , DNA , DNA/genética
8.
Front Cell Infect Microbiol ; 12: 863712, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35967845

RESUMO

Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Several agents that block cell division have been shown to mislocalise FtsZ, including the bacteriophage λ-encoded Kil peptide, resulting in defective cell division and a filamentous phenotype, making FtsZ an attractive target for antimicrobials. In this study, we have used an in vitro meningitis model system for studying the effect of bacteriophages on FtsZ using fluorescent E. coli EV36/FtsZ-mCherry and K12/FtsZ-mNeon strains. We show localisation of FtsZ to the bacterial cell midbody as a single ring during normal growth conditions, and mislocalisation of FtsZ producing filamentous multi-ringed bacterial cells upon addition of the known inhibitor Kil peptide. We also show that when bacteriophages K1F-GFP and T7-mCherry were applied to their respective host strains, these phages can inhibit FtsZ and block bacterial cell division leading to a filamentous multi-ringed phenotype, potentially delaying lysis and increasing progeny number. This occurs in the exponential growth phase, as actively dividing hosts are needed. We present that ZapA protein is needed for phage inhibition by showing a phenotype recovery with a ZapA mutant strain, and we show that FtsI protein is also mislocalised upon phage infection. Finally, we show that the T7 peptide gp0.4 is responsible for the inhibition of FtsZ in K12 strains by observing a phenotype recovery with a T7Δ0.4 mutant.


Assuntos
Proteínas de Bactérias , Bacteriófagos , Proteínas de Transporte , Proteínas do Citoesqueleto , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo
9.
Bioengineered ; 13(7-12): 14947-14959, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37105766

RESUMO

During the last decades, we have witnessed unprecedented advances in biological engineering and synthetic biology. These disciplines aim to take advantage of gene pathway regulation and gene expression in different organisms, to enable cells to perform desired functions. Yeast has been widely utilized as a model for the study of eukaryotic protein expression while bacteriophage T7RNAP and its promoter constitute the preferred system for prokaryotic protein expression (such as pET-based expression systems). The ability to integrate a T7RNAP-based expression system in yeast could allow for a better understanding of gene regulation in eukaryotic cells, and potentially increase the efficiency and processivity of yeast as an expression system. However, the attempts for the creation of such a system have been unsuccessful to date. This review aims to: (i) summarize the efforts that, for many years, have been devoted to the creation of a T7RNAP-based yeast expression system and ii) provide an overview of the latest advances in knowledge of eukaryotic transcription and translation that could lead to the construction of a successful T7RNAP expression system in yeast. The completion of this new expression system would allow to further expand the toolkit of yeast in synthetic biology and ultimately contribute to boost yeast usage as a key cell factory in sustainable biorefinery and circular economy.


Assuntos
RNA Polimerases Dirigidas por DNA , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Virais , Regiões Promotoras Genéticas/genética
10.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34074772

RESUMO

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.


Assuntos
Bacteriófagos/genética , Bacteriófagos/fisiologia , DNA/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Pontos de Checagem do Ciclo Celular , Divisão Celular , Endonucleases , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Uracila/metabolismo
12.
Nucleic Acids Res ; 48(10): 5397-5406, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32338761

RESUMO

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.


Assuntos
Bacteriófago T7/fisiologia , Proteínas Virais/metabolismo , Bacteriófago T7/genética , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/virologia , Plasmídeos , Proteínas Virais/genética
13.
Sci Rep ; 10(1): 307, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941920

RESUMO

Bacteriophage T7 is an intracellular parasite that recognizes its host via its tail and tail fiber proteins, known as receptor-binding proteins (RBPs). The RBPs attach to specific lipopolysaccharide (LPS) features on the host. Various studies have shown expansion of the phage's host range via mutations in the genes encoding the RBPs, whereas only a few have shown contraction of its host range. Furthermore, most experimental systems have not monitored the alteration of host range in the presence of several hosts simultaneously. Here we show that T7 phage grown in the presence of five restrictive strains and one permissive host, each with a different LPS form, gradually avoids recognition of the restrictive strains. Remarkably, avoidance of the restrictive strains was repeated in different experiments using six different permissive hosts. The evolved phages carried mutations that changed their specificity, as determined by sequencing of the genes encoding the RBPs. This system demonstrates a major role for RBPs in narrowing the range of futile infections. The system can be harnessed for host-range contraction in applications such as detection or elimination of a specific bacterial serotype by bacteriophages.


Assuntos
Bacteriófago T7/metabolismo , Evolução Molecular , Especificidade de Hospedeiro , Bacteriófago T7/patogenicidade , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
EMBO Rep ; 20(8): e48269, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31267640

RESUMO

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.


Assuntos
Cromossomos de Mamíferos , Edição de Genes/métodos , Genoma , Processos de Determinação Sexual , Razão de Masculinidade , Animais , Cruzamento , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cruzamentos Genéticos , Feminino , Fertilização , Masculino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo
15.
Cancer Immunol Res ; 7(3): 388-400, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30665890

RESUMO

Immunotherapies targeting T lymphocytes are revolutionizing cancer therapy but only benefit a subset of patients, especially in colorectal cancer. Thus, additional insight into the tumor microenvironment (TME) is required. Eosinophils are bone marrow-derived cells that have been largely studied in the context of allergic diseases and parasite infections. Although tumor-associated eosinophilia has been described in various solid tumors including colorectal cancer, knowledge is still missing regarding eosinophil activities and even the basic question of whether the TME promotes eosinophil recruitment without additional manipulation (e.g., immunotherapy) is unclear. Herein, we report that eosinophils are recruited into developing tumors during induction of inflammation-induced colorectal cancer and in mice with the Apcmin /+ genotype, which develop spontaneous intestinal adenomas. Using adoptive transfer and cytokine neutralization experiments, we demonstrate that the TME supported prolonged eosinophil survival independent of IL5, an eosinophil survival cytokine. Tumor-infiltrating eosinophils consisted of degranulating eosinophils and were essential for tumor rejection independently of CD8+ T cells. Transcriptome and proteomic analysis revealed an IFNγ-linked signature for intratumoral eosinophils that was different from that of macrophages. Our data establish antitumorigenic roles for eosinophils in colorectal cancer. These findings may facilitate the development of pharmacologic treatments that could unleash antitumor responses by eosinophils, especially in colorectal cancer patients displaying eosinophilia.


Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Eosinófilos/imunologia , Animais , Degranulação Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Quimiocina CCL11/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Imunoterapia Adotiva , Interferon gama/metabolismo , Interferon gama/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteômica , Transdução de Sinais , Microambiente Tumoral/imunologia
16.
RNA Biol ; 16(4): 595-599, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30146918

RESUMO

We recently developed a platform where phage-transducing particles optimize DNA delivery to a wide range of hosts. Here, we use this platform to optimize DNA transduction into hosts that naturally restrict specific DNA sequences. We first show that a specific plasmid is restricted for transduction into a particular Salmonella strain. Using the platform, we select for a mutated plasmid that overcomes the restriction barrier. Insertion of the non-mutated sequence into a permissive plasmid restricts transduction. We further show that epigenetic modification enables the DNA to evade restriction by the putative defense system. Our results validate this straightforward genetic approach for optimization of DNA transduction into new hosts.


Assuntos
DNA Bacteriano/genética , Evasão da Resposta Imune/genética , Mutação/genética , Transdução Genética , Sequência de Bases , Epigênese Genética , Plasmídeos/genética , Reprodutibilidade dos Testes
17.
Proc Natl Acad Sci U S A ; 115(23): E5353-E5362, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29789383

RESUMO

T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófago T7/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Escherichia coli/virologia , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , Transcrição Gênica
18.
J Cell Biochem ; 119(2): 1291-1298, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28731201

RESUMO

Generating plants with increased yields while maintaining low production and maintenance costs is highly important since plants are the major food source for humans and animals, as well as important producers of chemicals, pharmaceuticals, and fuels. Gene editing approaches, particularly the CRISPR-Cas system, are the preferred methods for improving crops, enabling quick, robust, and accurate gene manipulation. Nevertheless, new breeds of genetically modified crops have initiated substantial debates concerning their biosafety, commercial use, and regulation. Here, we discuss the challenges facing genetic engineering of crops by CRISPR-cas, and highlight the pros and cons of using this tool.


Assuntos
Sistemas CRISPR-Cas , Produtos Agrícolas , Alimentos Geneticamente Modificados , Edição de Genes/métodos , Legislação sobre Alimentos , Plantas Geneticamente Modificadas , Animais , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Humanos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
19.
Science ; 357(6356): 1096-1097, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28912231
20.
Bio Protoc ; 7(15)2017 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-28804739

RESUMO

We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages. This protocol allows seamless deletion of desired genes in a T7 phage, and can be expanded to other phages and other types of genetic manipulations as well.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...