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1.
Int J Mol Sci ; 21(19)2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998303

RESUMO

Some years inspire more hindsight reflection and future-gazing than others. This is even more so in 2020 with its evocation of perfect vision and the landmark ring to it. However, no futurist can reliably predict what the world will look like the next time that a year's first two digits will match the second two digits-a numerical pattern that only occurs once in a century. As we leap into a new decade, amid uncertainties triggered by unforeseen global events-such as the outbreak of a worldwide pandemic, the accompanying economic hardship, and intensifying geopolitical tensions-it is important to note the blistering pace of 21st century technological developments indicate that while hindsight might be 20/20, foresight is 50/50. The history of science shows us that imaginative ideas, research excellence, and collaborative innovation can, for example, significantly contribute to the economic, cultural, social, and environmental recovery of a post-COVID-19 world. This article reflects on a history of yeast research to indicate the potential that arises from advances in science, and how this can contribute to the ongoing recovery and development of human society. Future breakthroughs in synthetic genomics are likely to unlock new avenues of impactful discoveries and solutions to some of the world's greatest challenges.


Assuntos
Surtos de Doenças/prevenção & controle , Engenharia Genética/métodos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Saccharomyces cerevisiae/classificação
2.
Molecules ; 25(18)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899754

RESUMO

The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.


Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Chlamydomonas reinhardtii/genética , Infecções por Coronavirus/tratamento farmacológico , Lectinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Polifenóis/farmacologia , Polissacarídeos/farmacologia , Antivirais/química , Antivirais/isolamento & purificação , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/patogenicidade , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/química , Cloroplastos/genética , Cloroplastos/metabolismo , Infecções por Coronavirus/prevenção & controle , Engenharia Genética/métodos , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Pandemias , Polifenóis/química , Polifenóis/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Vacinas Virais/biossíntese , Vacinas Virais/farmacologia
3.
Nat Commun ; 11(1): 4418, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887885

RESUMO

Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.


Assuntos
Arabidopsis/genética , Cromossomos de Plantas , Engenharia Genética/métodos , Recombinação Genética , Sistemas CRISPR-Cas , Inversão Cromossômica , Troca Genética , Melhoramento Vegetal/métodos , Plantas
4.
Infez Med ; 28(3): 302-311, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32920565

RESUMO

SARS-CoV-2 has created a global disaster by infecting millions of people and causing thousands of deaths across hundreds of countries. Currently, the infection is in its exponential phase in several countries and there is no sign of immediate relief from this deadly virus. At the same time, some "conspiracy theories" have arisen on the origin of this virus due to the lack of a "definite origin". To understand if this controversy is also reflected in scientific publications, here, we reviewed the key articles published at initial stages of the COVID-19 pandemic (January 01, 2020 to April 30, 2020) related to the zoonotic origin of SARS-CoV-2 and the articles opposing the "conspiracy theories". We also provide an overview on the current knowledge on SARS-CoV-2 Spike as well as the Coronavirus research domain. Furthermore, a few important points related to the "conspiracy theories" such as "laboratory engineering" or "bioweapon" aspects of SARS-CoV-2 are also reviewed. In this article, we have only considered the peer-reviewed publications that are indexed in PubMed and other official publications, and we have directly quoted the authors' statements from their respective articles to avoid any controversy.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Engenharia Genética/métodos , Pneumonia Viral/virologia , Seleção Genética , Animais , Derramamento de Material Biológico , Armas Biológicas , Quirópteros/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Dissidências e Disputas , Eutérios/classificação , Eutérios/virologia , Saúde Global/estatística & dados numéricos , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Recombinação Genética , Alinhamento de Sequência , Zoonoses/virologia
5.
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
6.
Nat Commun ; 11(1): 4468, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901021

RESUMO

Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. Here, we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. EGI couples a dominant lethal transgene with a recessive resistance allele. Strains homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. EGI genotypes can also be tuned to cause hybrid lethality at different developmental life-stages. Further, we demonstrate that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. EGI is a simple and robust approach in multiple sexually reproducing organisms.


Assuntos
Drosophila melanogaster/genética , Engenharia Genética/métodos , Especiação Genética , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Feminino , Genes de Insetos , Genes Letais , Genótipo , Hibridização Genética , Masculino , Modelos Genéticos , Transgenes
7.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1269-1276, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748584

RESUMO

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.


Assuntos
Adenovírus Humanos , Vetores Genéticos , Terapia Viral Oncolítica , Vírus Oncolíticos , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Engenharia Genética/métodos , Engenharia Genética/tendências , Humanos , Terapia Viral Oncolítica/tendências , Vírus Oncolíticos/genética , Replicação Viral
8.
Nat Commun ; 11(1): 3847, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737299

RESUMO

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.


Assuntos
Aptâmeros de Nucleotídeos/genética , Arabidopsis/genética , Brassica/genética , Engenharia Genética/métodos , RNA Mensageiro/genética , Tabaco/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Arabidopsis/metabolismo , Compostos de Benzil/química , Brassica/metabolismo , Fluorescência , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Genes Reporter , Imidazolinas/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/metabolismo , Tabaco/metabolismo , Transformação Genética
9.
PLoS One ; 15(8): e0237675, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797066

RESUMO

RNA interference (RNAi), a technique used to investigate gene function in insects and other organisms, is attracting attention as a potential new technology for mosquito control. Saccharomyces cerevisiae (baker's yeast) was recently engineered to produce interfering RNA molecules that silence genes required for mosquito survival, but which do not correspond to genes in humans or other non-target organisms. The resulting yeast pesticides, which facilitate cost-effective production and delivery of interfering RNA to mosquito larvae that eat the yeast, effectively kill mosquitoes in laboratory and semi-field trials. In preparation for field evaluation of larvicides in Trinidad, a Caribbean island with endemic diseases resulting from pathogens transmitted by Aedes mosquitoes, adult residents living in the prospective trial site communities of Curepe, St. Augustine, and Tamana were engaged. Open community forums and paper surveys were used to assess the potential acceptability, societal desirability, and sustainability of yeast interfering RNA larvicides. These assessments revealed that Trinidadians have good working knowledge of mosquitoes and mosquito-borne illnesses. A majority of the respondents practiced some method of larval mosquito control and agreed that they would use a new larvicide if it were proven to be safe and effective. During the community engagement forums, participants were educated about mosquito biology, mosquito-borne diseases, and the new yeast larvicides. When invited to provide feedback, engagement forum attendees were strongly supportive of the new technology, raised few concerns, and provided helpful advice regarding optimal larvicide formulations, insecticide application, operational approaches for using the larvicides, and pricing. The results of these studies suggest that the participants are supportive of the potential use of yeast interfering RNA larvicides in Trinidad and that the communities assessed in this investigation represent viable field sites.


Assuntos
Aedes/genética , Engenharia Genética/métodos , Controle de Mosquitos/métodos , Interferência de RNA , Saccharomyces cerevisiae/genética , Adulto , Animais , Feminino , Humanos , Larva/genética , Masculino , Pessoa de Meia-Idade , Mosquitos Vetores/genética , Controle Biológico de Vetores/métodos , RNA Interferente Pequeno/genética , Características de Residência , Inquéritos e Questionários , Trinidad e Tobago
10.
PLoS One ; 15(7): e0232915, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706785

RESUMO

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell-impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.


Assuntos
Engenharia Genética/métodos , Regiões Promotoras Genéticas/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Lentivirus/genética , Células MCF-7 , RNA Mensageiro/genética , Transgenes/genética
11.
Genet Sel Evol ; 52(1): 35, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611306

RESUMO

Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecBB) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecBB mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Fertilidade/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/fisiologia , Animais , Cruzamento , Feminino , Engenharia Genética/métodos , Genótipo , Heterozigoto , Tamanho da Ninhada de Vivíparos/genética , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Gravidez , Ovinos/genética
12.
PLoS One ; 15(6): e0233911, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479550

RESUMO

Promoters are very important for transcriptional regulation and gene expression, and have become invaluable tools for genetic engineering. Owing to the characteristics of obligate biotrophs, molecular research into obligate biotrophic fungi is seriously lagging behind, and very few of their endogenous promoters have been developed. In this study, a WY7 fragment was predicted in the genome of Oidium heveae Steinmann using PromoterScan. Its promoter function was verified with transient transformations (Agrobacterium tumefaciens-mediated transformation, ATMT) in Nicotiana tabacum cv. Xanthi nc. The analysis of the transcription range showed that WY7 could regulate GUS expression in both monocots (Zea mays Linn and Oryza sativa L. spp. Japonica cv. Nipponbare) and dicots (N. tabacum and Hylocereus undulates Britt). The results of the quantitative detection showed that the GUS transient expression levels when regulated by WY7 was more than 11.7 times that of the CaMV 35S promoter in dicots (N. tabacum) and 5.13 times that of the ACT1 promoter in monocots (O. sativa). GUS staining was not detected in the T1 generation of the WY7-GUS transgenic N. tabacum. This showed that WY7 is an inducible promoter. The cis elements of WY7 were predicted using PlantCARE, and further experiments indicated that WY7 was a low temperature- and salt-inducible promoter. Soluble proteins produced by WY7-hpa1Xoo transgenic tobacco elicited hypersensitive responses (HR) in N. tabacum leaves. N. tabacum transformed with pBI121-WY7-hpa1Xoo exhibited enhanced resistance to the tobacco mosaic virus (TMV). The WY7 promoter has a lot of potential as a tool for plant genetic engineering. Further in-depth studies will help to better understand the transcriptional regulation mechanisms of O. heveae.


Assuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Doenças das Plantas/prevenção & controle , Regiões Promotoras Genéticas , Fungos/patogenicidade , Genoma Fúngico , Hevea/genética , Hevea/microbiologia , Interações Hospedeiro-Patógeno/genética , Magnoliopsida/genética , Magnoliopsida/microbiologia , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Tabaco/genética , Tabaco/microbiologia , Transformação Genética , Zea mays/genética , Zea mays/microbiologia
13.
Nat Commun ; 11(1): 2739, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483165

RESUMO

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.


Assuntos
Escherichia coli/imunologia , Imunoterapia/métodos , Proteínas de Membrana/imunologia , Neoplasias/terapia , Transdução de Sinais/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Fagócitos/imunologia , Fagócitos/metabolismo , Transdução de Sinais/genética , Biologia Sintética/métodos , Biologia Sintética/tendências
14.
Int J Biol Macromol ; 160: 736-740, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32485251

RESUMO

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic in the past four months and causes respiratory disease in humans of almost all ages. Although several drugs have been announced to be partially effective treatments for this disease, no approved vaccine is available. Here, we described the construction of a recombinant Lactobacillus plantarum strain expressing the SARS-CoV-2 spike protein. The results showed that the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity. The highest protein yield was obtained under the following conditions: cells were induced with 50 ng/mL SppIP at 37 °C for 6-10 h. The recombinant spike (S) protein was stable under normal conditions and at 50 °C, pH = 1.5, or a high salt concentration. Recombinant L. plantarum may provide a promising food-grade oral vaccine candidate against SARS-CoV-2 infection.


Assuntos
DNA Recombinante/genética , Engenharia Genética/métodos , Lactobacillus plantarum/genética , Glicoproteína da Espícula de Coronavírus/genética , Expressão Gênica
15.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510483

RESUMO

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.


Assuntos
Engenharia Genética/métodos , Ensaios de Triagem em Larga Escala/métodos , RNA Fúngico/análise , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA/métodos , Esferoplastos/genética , Fenótipo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo
16.
Nat Commun ; 11(1): 2427, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415193

RESUMO

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.


Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Oxirredução , Eletroquímica , Eletrodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferricianetos/química , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Piocianina/química , Percepção de Quorum , Regulon , Salmonella enterica/metabolismo , Espectrometria de Fluorescência
17.
Mol Cell ; 78(4): 614-623, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32442504

RESUMO

Synthetic biology has promised and delivered on an impressive array of applications based on genetically modified microorganisms. While novel biotechnology undoubtedly offers benefits, like all new technology, precautions should be considered during implementation to reduce the risk of both known and unknown adverse effects. To achieve containment of transgenic microorganisms, confidence to a near-scientific certainty that they cannot transfer their transgenic genes to other organisms, and that they cannot survive to propagate in unintended environments, is a priority. Here, we present an in-depth summary of biological containment systems for micro-organisms published to date, including the production of a genetic firewall through genome recoding and physical containment of microbes using auxotrophies, regulation of essential genes, and expression of toxic genes. The level of containment required to consider a transgenic organism suitable for deployment is discussed, as well as standards of practice for developing new containment systems.


Assuntos
Biotecnologia/métodos , Genes Essenciais , Engenharia Genética/métodos , Microbiota/genética , Microrganismos Geneticamente Modificados/genética , Biologia Sintética/métodos , Humanos , Microrganismos Geneticamente Modificados/crescimento & desenvolvimento
18.
PLoS One ; 15(4): e0232046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352996

RESUMO

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.


Assuntos
Engenharia Genética/métodos , RNA Catalítico/fisiologia , Biologia Sintética/métodos , Animais , Drosophila/genética , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Expressão Gênica/genética , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Conformação de Ácido Nucleico , Estudo de Prova de Conceito , RNA Catalítico/genética , RNA Mensageiro/metabolismo
19.
Gene ; 753: 144795, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32450202

RESUMO

The advent of genetic selection and genome modification method assure about a real novel reformation in biotechnology and genetic engineering. With the extensive capabilities of molecular markers of them being stable, cost-effective and easy to use, they ultimately become a potent tool for variety of applications such a gene targeting, selection, editing, functional genomics; mainly for the improvisation of commercially important crops. Three main benefits of molecular marker in the field of agriculture and crop improvement programmes first, reduction of the duration of breeding programmes, second, they allow creation of new genetic variation and genetic diversity of plants and third most promising benefit is help in production of engineered plant for disease resistance, or resistance from pathogen and herbicides. This review is anticipated to present an outline how the techniques have been evolved from the simple conventional applications of DNA based molecular markers to highly throughput CRISPR technology and geared the crop yield. Techniques like using Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems have revolutionised in the field of genome editing. These have been promptly accepted in both the research and commercial industry. On the whole, the widespread use of molecular markers with their types, their appliance in plant breeding along with the advances in genetic selection and genome editing together being a novel strategy to boost crop yield has been reviewed.


Assuntos
Agricultura/métodos , Produtos Agrícolas/genética , Engenharia Genética/métodos , Biomarcadores , Biotecnologia , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Resistência à Doença/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Genoma de Planta/genética , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética
20.
Nat Commun ; 11(1): 2106, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355156

RESUMO

Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.


Assuntos
Drosophila melanogaster/efeitos dos fármacos , Engenharia Genética/métodos , Gentamicinas/farmacologia , Puromicina/farmacologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Resistência a Medicamentos , Éxons , Feminino , Genética Populacional , Homozigoto , Íntrons , Masculino , Controle de Pragas , Processamento de RNA , Análise para Determinação do Sexo
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