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1.
Life Sci ; 256: 117911, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504756

RESUMO

AIMS: To explore the potential regulatory mechanism of differentially expressed mRNAs in Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). MAIN METHODS: Patients with HCV-related HCC and age- and gender-matched healthy subjects were enrolled. Differentially expressed mRNAs in the plasma were detected by digital gene expression (DGE) profile analysis. HepG2 and SMMC7721 cells stably transfected with HCV-core protein and the control plasmid were established. And small interfering RNA (siRNA) was used to knockdown the target gene in HCV core-expressing HCC cell lines. mRNA expression was determined by qRT-PCR. Protein expression was measured by Western blot and immunohistochemistry staining. KEY FINDINGS: DGE profile data showed aberrant mRNA expression contributed to the progression of HCV-HCC, and clusterin (CLU), which was significantly highly expressed, was chosen as a candidate gene. Further evidence showed CLU was highly expressed in tumor tissues of HCV-HCC patients and HCV core-expressing HCC cell lines, accompanied with enhanced autophagy and upregulation of pro-autophagy genes. And knockdown of CLU in HCC cell lines suppressed cell autophagy, which was indicated by decreased expression of autophagy marker light chain 3B (LC3B) ІІ/І ratio, and downregulated pro-autophagy genes like Beclin1, autophagy-related protein 7 (Atg7) and Lamp2. On the other hand, anti-autophagy genes or regulators, including p62 and phosphorylated mammalian target of rapamycin (p-mTOR), were notably upregulated. SIGNIFICANCE: CLU could promote the progression of HCV-related HCC by regulating autophagy, which might be a potential therapeutic target of HCV-HCC.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Clusterina/metabolismo , Hepacivirus/metabolismo , Neoplasias Hepáticas/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Biblioteca Genômica , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , RNA Mensageiro/efeitos dos fármacos , RNA Interferente Pequeno/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
2.
Cell ; 180(5): 1002-1017.e31, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109417

RESUMO

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.


Assuntos
Sistemas CRISPR-Cas/genética , Genoma Bacteriano/genética , Biblioteca Genômica , Staphylococcus aureus/genética , Escherichia coli/genética , Humanos , RNA Bacteriano/genética , RNA Guia/genética , Streptococcus pyogenes/genética
3.
Nucleic Acids Res ; 48(8): e48, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32095820

RESUMO

Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.


Assuntos
Vias Biossintéticas/genética , Metagenômica/métodos , Técnicas Analíticas Microfluídicas , Biblioteca Genômica , Dispositivos Lab-On-A-Chip , Plasmídeos/genética , Policetídeo Sintases/genética , Microbiologia do Solo , Fluxo de Trabalho
4.
Nat Biotechnol ; 38(3): 355-364, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932729

RESUMO

A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.


Assuntos
Biologia Computacional/métodos , Expressão Gênica , RNA Guia/genética , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Aprendizado Profundo , Edição de Genes , Biblioteca Genômica , Células HeLa , Humanos , Células K562 , Fenótipo , Análise de Sequência de RNA
6.
Nat Commun ; 10(1): 5794, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31857575

RESUMO

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype-phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.


Assuntos
Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico/métodos , Genoma Fúngico/genética , Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Biotecnologia/métodos , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Biblioteca Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Fenótipo , Saccharomyces cerevisiae/genética
7.
PLoS Genet ; 15(11): e1008397, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31693674

RESUMO

In animals, circadian rhythms are driven by oscillations in transcription, translation, and proteasomal degradation of highly conserved genes, resulting in diel cycles in the expression of numerous clock-regulated genes. Transcription is largely regulated through the binding of transcription factors to cis-regulatory elements within accessible regions of the chromatin. Chromatin remodeling is linked to circadian regulation in mammals, but it is unknown whether cycles in chromatin accessibility are a general feature of clock-regulated genes throughout evolution. To assess this, we applied an ATAC-seq approach using Nematostella vectensis, grown under two separate light regimes (light:dark (LD) and constant darkness (DD)). Based on previously identified N. vectensis circadian genes, our results show the coupling of chromatin accessibility and circadian transcription rhythmicity under LD conditions. Out of 180 known circadian genes, we were able to list 139 gene promoters that were highly accessible compared to common promoters. Furthermore, under LD conditions, we identified 259 active enhancers as opposed to 333 active enhancers under DD conditions, with 171 enhancers shared between the two treatments. The development of a highly reproducible ATAC-seq protocol integrated with published RNA-seq and ChIP-seq databases revealed the enrichment of transcription factor binding sites (such as C/EBP, homeobox, and MYB), which have not been previously associated with circadian signaling in cnidarians. These results provide new insight into the regulation of cnidarian circadian machinery. Broadly speaking, this supports the notion that the association between chromatin remodeling and circadian regulation arose early in animal evolution as reflected in this non-bilaterian lineage.


Assuntos
Ritmo Circadiano/genética , Cnidários/genética , Elementos Facilitadores Genéticos/genética , Transcrição Genética , Animais , Cromatina/genética , Relógios Circadianos/genética , Cnidários/crescimento & desenvolvimento , Escuridão , Regulação da Expressão Gênica no Desenvolvimento/genética , Biblioteca Genômica , Fotoperíodo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética
8.
Genome Biol Evol ; 11(12): 3353-3371, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31702783

RESUMO

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.


Assuntos
Cromossomos de Plantas/genética , Ericaceae/genética , Genoma de Planta/genética , Rhododendron/genética , Sintenia , Sequência de Bases , Cromatina/genética , Mapeamento Cromossômico , Ligação Genética , Biblioteca Genômica , Anotação de Sequência Molecular , Transposases/genética
9.
Genome Biol ; 20(1): 226, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672156

RESUMO

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing.


Assuntos
Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica/métodos , Animais , Benchmarking , Microbioma Gastrointestinal , Humanos , Camundongos
10.
Clin Chem ; 65(12): 1581-1591, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31645340

RESUMO

BACKGROUND: Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS: We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5'-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5'-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3'-phosphorylated RNAs to enter the library. RESULTS: Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS: Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.


Assuntos
Técnicas de Química Combinatória/métodos , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Viés , DNA Complementar , Biblioteca Gênica , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs , RNA Interferente Pequeno , RNA de Transferência , Análise de Sequência de RNA/métodos
11.
Reprod Biol Endocrinol ; 17(1): 85, 2019 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-31656198

RESUMO

BACKGROUND: Voluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility. METHODS: Genomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-α sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg++-dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice. RESULTS: The 80 kDa rSAF showed complete sperm agglutination, inhibited its Mg2+-ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests. CONCLUSION: The Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.


Assuntos
Colo do Útero/microbiologia , Anticoncepcionais Femininos/farmacologia , Aglutinação Espermática/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Staphylococcus/metabolismo , Acrossomo/efeitos dos fármacos , Acrossomo/fisiologia , Animais , Anticoncepcionais Femininos/metabolismo , Feminino , Biblioteca Genômica , Humanos , Infertilidade Feminina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Staphylococcus/genética
12.
Methods Mol Biol ; 2024: 181-198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31364050

RESUMO

In the last two decades, phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Streptococcus pneumoniae-and the use of this library for the molecular dissection of the human B-cell response against bacterial infection and colonization.


Assuntos
Biblioteca de Peptídeos , Streptococcus pneumoniae/imunologia , Antígenos/imunologia , Bacteriófago lambda/metabolismo , DNA Complementar/metabolismo , Biblioteca Genômica
13.
Nat Commun ; 10(1): 3583, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395865

RESUMO

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.


Assuntos
Biologia Computacional/métodos , Doença/genética , Mutagênese , Elementos Reguladores de Transcrição/genética , Linhagem Celular , Clonagem Molecular , Genoma Humano/genética , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único
14.
PLoS Comput Biol ; 15(8): e1007273, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31433799

RESUMO

Long-read sequencing and novel long-range assays have revolutionized de novo genome assembly by automating the reconstruction of reference-quality genomes. In particular, Hi-C sequencing is becoming an economical method for generating chromosome-scale scaffolds. Despite its increasing popularity, there are limited open-source tools available. Errors, particularly inversions and fusions across chromosomes, remain higher than alternate scaffolding technologies. We present a novel open-source Hi-C scaffolder that does not require an a priori estimate of chromosome number and minimizes errors by scaffolding with the assistance of an assembly graph. We demonstrate higher accuracy than the state-of-the-art methods across a variety of Hi-C library preparations and input assembly sizes. The Python and C++ code for our method is openly available at https://github.com/machinegun/SALSA.


Assuntos
Cromossomos Humanos/genética , Genoma Humano , Genômica/métodos , Algoritmos , Animais , Biologia Computacional , Simulação por Computador , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Biblioteca Genômica , Genômica/estatística & dados numéricos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/estatística & dados numéricos , Software
15.
Mol Plant Microbe Interact ; 32(12): 1564-1570, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31272284

RESUMO

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.


Assuntos
Ascomicetos , Genoma Fúngico , Biblioteca Genômica , Genômica , Fases de Leitura Aberta , Ascomicetos/genética , Genoma Fúngico/genética , Genômica/métodos , Fases de Leitura Aberta/genética , Triticum/microbiologia
16.
Genes (Basel) ; 10(7)2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31248052

RESUMO

The Steller sea lion is the largest member of the Otariidae family and is found in the coastal waters of the northern Pacific Rim. Here, we present the Steller sea lion genome, determined through DNA sequencing approaches that utilized microfluidic partitioning library construction, as well as nanopore technologies. These methods constructed a highly contiguous assembly with a scaffold N50 length of over 14 megabases, a contig N50 length of over 242 kilobases and a total length of 2.404 gigabases. As a measure of completeness, 95.1% of 4104 highly conserved mammalian genes were found to be complete within the assembly. Further annotation identified 19,668 protein coding genes. The assembled genome sequence and underlying sequence data can be found at the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA475770.


Assuntos
Genoma , Leões-Marinhos/genética , Animais , Biblioteca Genômica , Microfluídica/métodos , Nanoporos , Sequenciamento Completo do Genoma
17.
Acta Virol ; 63(2): 129-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230441

RESUMO

The integrated proviral genome is the major barrier to a cure for HIV-1 infection. Genome editing technologies, such as CRISPR/Cas9, may disable or remove the HIV-1 provirus by introducing DNA double strand breaks at sequence specific sites in the viral genome. Host DNA repair by the error-prone non-homologous end joining pathway generates mutagenic insertions or deletions at the break. CRISPR/Cas9 editing has been shown to reduce replication competent viral genomes in cell culture, but only a minority of possible genome editing targets have been assayed. Currently there is no map of double strand break genetic fitness for HIV-1 to inform the choice of editing targets. However, CRISPR/Cas9 genome editing makes it possible to target double strand breaks along the length of the provirus to generate a double strand break genetic fitness map. We identified all possible HIV-1 targets with different bacterial species of CRISPR/Cas9. This library of guide RNAs was evaluated for GC content and potential off-target sites in the human genome. Complexity of the library was reduced by eliminating duplicate guide RNA targets in the HIV-1 long terminal repeats and targets in the env gene. Although the HIV-1 genome is AT-rich, the S. pyogenes CRISPR/Cas9 with the proto-spacer adjacent motif NGG offers the most HIV-1 guide RNAs. This library of HIV-1 guide RNAs may be used to generate a double strand break genetic fragility map to be further applied to any genome editing technology designed for the HIV-1 provirus. Keywords: HIV-1; genome editing; CRISPR; genetic fitness; guide RNAs.


Assuntos
Aptidão Genética , Biblioteca Genômica , HIV-1 , Sistemas CRISPR-Cas , Genoma Viral , HIV-1/genética , Humanos , Provírus/genética
18.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213554

RESUMO

Host-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogen Streptococcus pneumoniae generated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism's transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was the dlt locus, which adds d-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence of d-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of the dlt locus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in a lysM-/- host, there was no longer an effect of the dlt locus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host.IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism's capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiased in vivo transposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by the dlt locus, previously shown to add d-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.


Assuntos
Proteínas de Bactérias/genética , Derrame de Bactérias , Inflamação , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/transmissão , Sistema Respiratório/microbiologia , Streptococcus pneumoniae/genética , Alanina/metabolismo , Animais , Animais Recém-Nascidos , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Biblioteca Genômica , Interações Hospedeiro-Patógeno/imunologia , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Streptococcus pneumoniae/imunologia , Ácidos Teicoicos/metabolismo
19.
Appl Microbiol Biotechnol ; 103(14): 5917-5923, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31111182

RESUMO

Aliphatic medium-chain alkanes, a major component of gasoline, diesel, and jet fuels, are drop-in compatible fuels. Microorganisms with the capacity to produce medium-chain alkanes are promising for the bio-production of drop-in fuel. We found that Klebsiella sp. NBRC100048 has the ability to produce medium-chain alkanes from medium-chain aldehydes. We cloned a gene involved in conversion of aldehydes to alkanes by using a genomic fosmid library derived from Klebsiella sp. NBRC100048. The gene termed orf2991 encodes 506 amino acids and shows 62% sequence homology to the aldehyde dehydrogenase of Escherichia coli, aldB. The finding of orf2991 as a novel alkane-synthesizing enzyme gene similar to E. coli aldehyde dehydrogenase family, which is generally known to catalyze a reaction oxidizing aldehydes to fatty acids, indicated a novel function of aldehyde dehydrogenase. This finding is not only significant academically but allows developing the novel manufacturing methods of alkanes fermentation.


Assuntos
Alcanos/metabolismo , Proteínas de Bactérias/genética , Klebsiella/genética , Aldeído Desidrogenase/genética , Aldeídos/metabolismo , Proteínas de Bactérias/metabolismo , Biocombustíveis , Clonagem Molecular , Escherichia coli/genética , Biblioteca Genômica , Klebsiella/metabolismo , Engenharia Metabólica , Homologia de Sequência
20.
Appl Microbiol Biotechnol ; 103(13): 5259-5267, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31069485

RESUMO

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.


Assuntos
Técnicas de Visualização da Superfície Celular , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriófago T7/genética , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Genoma Bacteriano , Biblioteca Genômica , Humanos , Immunoblotting , Lactente , Pessoa de Meia-Idade , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Testes Sorológicos , Tuberculose/sangue , Adulto Jovem
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