Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Methods Mol Biol ; 2110: 139-149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32002907

RESUMO

The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.

2.
Methods Mol Biol ; 2075: 399-409, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31584178

RESUMO

Conjugative assembly genome engineering (CAGE) enables the transfer of large chromosomal regions from a donor to a recipient. Specific regions of the donor chromosome can be introduced in the recipient genome by the directed insertion of an origin of transfer and two selection cassettes. Multiple paired CAGE experiments can be combined to generate chimeric chromosomes from different donor strains.

3.
Rev. bioét. derecho ; (47): 5-15, nov. 2019. ilus
Artigo em Inglês | IBECS | ID: ibc-184862

RESUMO

DNA read and write technologies have accelerated biotechnology at an unprecedented pace. This enhanced capacity to engineer living beings has accelerated not only scientific research, but also the translation into novel therapies. New approved medicinal products include the correction of the diseased genome and synthetic enhancement to fight diseases. These practices are widely supported socially and scientifically. Applications beyond therapy have also be attempted. In 2018, researcher He Jiankui reported on the edition of human germline during the Second International Summit on Human Genome Editing. On the other hand, during the last years, there have also been attempts at somatic genetic enhancement without the provision of detailed outcomes. Reading and writing DNA empowers us to change our world, even ourselves. The social benefits may be enormous. We need to accelerate the debate, including the stakeholders, to foster a responsible use of these technologies and maximize the positive impact on society


Las tecnologías de lectura y escritura de ADN han acelerado la biotecnología a un ritmo sin precedentes. Esta capacidad mejora para diseñar seres vivos no solo ha acelerado la investigación científica, sino también la translación a terapias novedosas. Nuevos medicamentos aprobados incluyen la corrección del genoma enfermo y la mejora sintética para combatir las enfermedades. Estas prácticas son ampliamente apoyadas social y científicamente. También se han intentado aplicaciones más allá de la terapia. En 2018, el investigador He Jiankui informó sobre la edición de la línea germinal humana durante la Segunda Cumbre Internacional sobre la Edición del Genoma Humano. Por otro lado, en los últimos años también se han producido intentos de mejora genética somática. Leer y escribir ADN nos permite cambiar nuestro planeta, incluso cambiarnos a nosotros mismos. Los beneficios sociales


Les tecnologies de lectura i escriptura d'ADN han accelerat la biotecnologia a un ritme sense precedents. Aquesta capacitat millorada per dissenyar éssers vius no només ha accelerat la recerca científica, sinó també la translació a teràpies noves. Nous medicaments aprovats inclouen la correcció del genoma malalt i la millora sintètica per a combatre les malalties. Aquestes pràctiques són àmpliament recolzades social i científicament. També s'han intentat aplicacions més enllà de la teràpia. El 2018, l'investigador He Jiankui va informar sobre l'edició de la línia germinal humana durant la Segona Cimera Internacional sobre l'Edició del Genoma Humà. D'altra banda, en els últims anys també s'han produït intents de millora genètica somàtica. Llegir i escriure ADN ens permet canviar el nostre planeta, fins i tot canviar-nos a nosaltres mateixos. Els beneficis socials poden ser enormes. Necessitem accelerar el debat, incloent-hi les parts interessades a fi de fomentar un ús responsable d'aquestes tecnologies i maximitzar-ne l'impacte positiu en la societat


Assuntos
Humanos , Edição de Genes , Pesquisa Médica Translacional , Biologia Sintética/ética , Biotecnologia/ética , Biotecnologia/instrumentação , Biotecnologia/legislação & jurisprudência , Avaliação da Tecnologia Biomédica/legislação & jurisprudência
5.
Acta Derm Venereol ; 99(13): 1253-1257, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573666

RESUMO

Imbalance in skin microflora, particularly related to certain Cutibacterium acnes strains, may trigger acne. Application of non-acne-causing strains to the skin may modulate the skin microbiome and thereby lead to a reduction in acne. This pilot study evaluates the safety and efficacy of microbiome modulation on acne-prone skin. The study had 2 phases: active induction (5% benzoyl peroxide gel, 7 days) and interventional C. acnes strains treatment (5 weeks). Patients were randomized to either topical skin formulations PT1 (2 strains of C. acnes Single Locus Sequence Typing [SLST] type C3 and K8, 50% each) or PT2 (4 strains of C. acnes SLST type C3 [55%], K8 [5%], A5 [30%] and F4 [10%]). Safety and efficacy was evaluated in 14 patients (PT1=8/14, PT2=6/14). Skin microbiome composition shifted towards study formulations. No untoward adverse events, visible irritation, or significant flare-up were observed. Non-inflamed lesions and skin pH were reduced. Comedone counts improved clinically with no deterioration in inflammatory lesions.

6.
Microbiome ; 7(1): 95, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234928

RESUMO

BACKGROUND: The skin is colonized by a large number of microorganisms, most of which are beneficial or harmless. However, disease states of skin have specific microbiome compositions that are different from those of healthy skin. Gut microbiome modulation through fecal transplant has been proven as a valid therapeutic strategy in diseases such as Clostridium difficile infections. Therefore, techniques to modulate the skin microbiome composition may become an interesting therapeutic option in diseases affecting the skin such as psoriasis or acne vulgaris. METHODS: Here, we have used mixtures of different skin microbiome components to alter the composition of recipient skin microbiomes. RESULTS: We show that after sequential applications of a donor microbiome, the recipient microbiome becomes more similar to the donor. After intervention, an initial week-long phase is characterized by the dominance of donor strains. The level of engraftment depends on the composition of the recipient and donor microbiomes, and the applied bacterial load. We observed higher engraftment using a multi-strain donor solution with recipient skin rich in Cutibacterium acnes subtype H1 and Leifsonia. CONCLUSIONS: We have demonstrated the use of living bacteria to modulate skin microbiome composition.

7.
Methods Mol Biol ; 1961: 3-11, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912036

RESUMO

Gene editing has great therapeutic impact, being of interest for many scientists worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has been adapted for gene editing to serve as an efficient, rapid, and cost-effective tool. To fulfill CRISPR experiment's goals, two components are important: an endonuclease and a gRNA. The most commonly used endonucleases are Cpf1 and Cas9 and are described in depth in this chapter. The gRNA targets the genome site to be edited, giving great importance to its design to obtain increased efficiency and decreased off-target events. In this chapter, we describe different tools to design suitable gRNAs for a variety of experimental purposes.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , RNA Guia/genética , Proteína 9 Associada à CRISPR/genética
8.
Biotechnol Prog ; 34(1): 271-276, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28960932

RESUMO

The establishment of erythromycin production within the heterologous host E. coli marked an accomplishment in genetic transfer capacity. Namely, over 20 genes and 50 kb of DNA was introduced to E. coli for successful heterologous biosynthetic reconstitution. However, the prospect for production levels that approach those of the native host requires the application of engineering tools associated with E. coli. In this report, metabolic and genomic engineering were implemented to improve the E. coli cellular background and the plasmid platform supporting heterologous erythromycin formation. Results include improved plasmid stability and metabolic support for biosynthetic product formation. Specifically, the new plasmid design for erythromycin formation allowed for ≥89% stability relative to current standards (20% stability). In addition, the new strain (termed LF01) designed to improve carbon flow to the erythromycin biosynthetic pathway provided a 400% improvement in titer level. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:271-276, 2018.


Assuntos
Eritromicina/biossíntese , Escherichia coli/genética , Engenharia Metabólica/métodos , Plasmídeos/genética , Vias Biossintéticas/genética , Carbono/metabolismo , Eritromicina/química , Genômica , Plasmídeos/biossíntese
10.
Prog Mol Biol Transl Sci ; 152: 95-114, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29150007

RESUMO

CRISPR-Cas9 has revolutionized the generation of transgenic animals. This system has demonstrated an unprecedented efficiency, multiplexability, and ease of use, thereby reducing the time and cost required for genome editing and enabling the production of animals with more extensive genetic modifications. It has also been shown to be applicable to a wide variety of animals, from early-branching metazoans to primates. Genome-wide screens in model organisms have been performed, accurate models of human diseases have been constructed, and potential therapies have been tested and validated in animal models. Several achievements in genetic modification of animals have been translated into products for the agricultural and pharmaceutical industries. Based on the remarkable progress to date, one may anticipate that in the future, CRISPR-Cas9 technology will enable additional far-reaching advances, including understanding the bases of diseases with complex genetic origins, engineering animals to produce organs for human transplantation, and genetically transforming entire populations of organisms to prevent the spread of disease.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais , Animais , Engenharia Genética , Terapia Genética
12.
Science ; 357(6357): 1303-1307, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28798043

RESUMO

Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concerns about pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. We previously demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. We now confirm that PERVs infect human cells, and we observe the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all of the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlights the value of PERV inactivation to prevent cross-species viral transmission and demonstrates the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.


Assuntos
Sistemas CRISPR-Cas , Transmissão de Doença Infecciosa/prevenção & controle , Retrovirus Endógenos/genética , Edição de Genes/métodos , Infecções por Retroviridae/transmissão , Sus scrofa/genética , Sus scrofa/virologia , Transplante Heterólogo/efeitos adversos , Animais , Células HEK293 , Humanos
13.
Nat Commun ; 7: 13330, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27804970

RESUMO

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.


Assuntos
Citidina Desaminase/genética , Edição de Genes , Engenharia Genética , Proteínas Recombinantes de Fusão/genética , Sequência de Bases , Desaminação , Escherichia coli/metabolismo , Genoma Humano , Células HEK293 , Humanos , Especificidade por Substrato
14.
Science ; 353(6301): 819-22, 2016 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-27540174

RESUMO

Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Sintéticos , Código Genético/fisiologia , Genoma Bacteriano , Genes Essenciais , Genes Letais , Código Genético/genética , Engenharia Genética , Fenótipo , Biossíntese de Proteínas/genética
15.
Science ; 350(6264): 1101-4, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26456528

RESUMO

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.


Assuntos
Retrovirus Endógenos/genética , Marcação de Genes/métodos , Infecções por Retroviridae/prevenção & controle , Suínos/virologia , Transplante Heterólogo/métodos , Inativação de Vírus , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Células Epiteliais/virologia , Dosagem de Genes , Genes pol , Células HEK293 , Humanos , Rim/virologia , Dados de Sequência Molecular , Infecções por Retroviridae/transmissão , Infecções por Retroviridae/virologia
16.
Bioinformatics ; 30(20): 2968-70, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24990609

RESUMO

SUMMARY: Clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies have revolutionized human genome engineering and opened countless possibilities to basic science, synthetic biology and gene therapy. Albeit the enormous potential of these tools, their performance is far from perfect. It is essential to perform a posterior careful analysis of the gene editing experiment. However, there are no computational tools for genome editing assessment yet, and current experimental tools lack sensitivity and flexibility. We present a platform to assess the quality of a genome editing experiment only with three mouse clicks. The method evaluates next-generation data to quantify and characterize insertions, deletions and homologous recombination. CRISPR Genome Analyzer provides a report for the locus selected, which includes a quantification of the edited site and the analysis of the different alterations detected. The platform maps the reads, estimates and locates insertions and deletions, computes the allele replacement efficiency and provides a report integrating all the information. AVAILABILITY AND IMPLEMENTATION: CRISPR-GA Web is available at http://crispr-ga.net. Documentation on CRISPR-GA instructions can be found at http://crispr-ga.net/documentation.html CONTACT: mguell@genetics.med.harvard.edu.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Engenharia Genética , Genômica/métodos , Alelos , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interface Usuário-Computador
17.
Nucleic Acids Res ; 41(19): 9049-61, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23907390

RESUMO

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.


Assuntos
Desoxirribonucleases/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Reparo Gênico Alvo-Dirigido/métodos , Linhagem Celular , Separação Celular , Desoxirribonucleases/química , Loci Gênicos , Genoma Humano , Humanos , Oligodesoxirribonucleotídeos , RNA Guia , Reparo de DNA por Recombinação
18.
Science ; 339(6121): 823-6, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23287722

RESUMO

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Marcação de Genes/métodos , Engenharia Genética/métodos , Genoma Humano , RNA/química , Cromossomos Humanos Par 19/genética , Códon/genética , Clivagem do DNA , Éxons , Loci Gênicos , Humanos , Células-Tronco Pluripotentes Induzidas , Sequências Repetidas Invertidas/genética , Células K562 , RNA/genética
19.
Mol Syst Biol ; 8: 585, 2012 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-22617959

RESUMO

Here, we report the genome-wide identification of small RNAs associated with transcription start sites (TSSs), termed tssRNAs, in Mycoplasma pneumoniae. tssRNAs were also found to be present in a different bacterial phyla, Escherichia coli. Similar to the recently identified promoter-associated tiny RNAs (tiRNAs) in eukaryotes, tssRNAs are associated with active promoters. Evidence suggests that these tssRNAs are distinct from previously described abortive transcription RNAs. ssRNAs have an average size of 45 bases and map exactly to the beginning of cognate full-length transcripts and to cryptic TSSs. Expression of bacterial tssRNAs requires factors other than the standard RNA polymerase holoenzyme. We have found that the RNA polymerase is halted at tssRNA positions in vivo, which may indicate that a pausing mechanism exists to prevent transcription in the absence of genes. These results suggest that small RNAs associated with TSSs could be a universal feature of bacterial transcription.


Assuntos
Escherichia coli/genética , Pneumonia por Mycoplasma/genética , RNA Bacteriano , Sítio de Iniciação de Transcrição , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Transcrição Genética
20.
Nat Rev Microbiol ; 9(9): 658-69, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21836626

RESUMO

Over the past 3 years, bacterial transcriptomics has undergone a massive revolution. Increased sequencing capacity and novel tools have made it possible to explore the bacterial transcriptome to an unprecedented depth, which has revealed that the transcriptome is more complex and dynamic than expected. Alternative transcripts within operons challenge the classic operon definition, and many small RNAs involved in the regulation of transcription, translation and pathogenesis have been discovered. Furthermore, mRNAs may localize to specific areas in the cell, and the spatial organization and dynamics of the chromosome have been shown to be important for transcription. Epigenetic modifications of DNA also affect transcription, and RNA processing affects translation. Therefore, transcription in bacteria resembles that in eukaryotes in terms of complexity more closely than was previously thought. Here we will discuss the contribution of 'omics' approaches to these discoveries as well as the possible impact that they are expected to have in the future.


Assuntos
Bactérias/genética , Perfilação da Expressão Gênica/métodos , RNA Bacteriano/genética , Epigênese Genética , Perfilação da Expressão Gênica/instrumentação , Regulação Bacteriana da Expressão Gênica , Óperon , Transcrição Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA