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Nat Biotechnol ; 37(11): 1380, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31591553


An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nat Biotechnol ; 37(10): 1115-1117, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31537915
BMC Med ; 17(1): 68, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30914045


Blockchain is a shared distributed digital ledger technology that can better facilitate data management, provenance and security, and has the potential to transform healthcare. Importantly, blockchain represents a data architecture, whose application goes far beyond Bitcoin - the cryptocurrency that relies on blockchain and has popularized the technology. In the health sector, blockchain is being aggressively explored by various stakeholders to optimize business processes, lower costs, improve patient outcomes, enhance compliance, and enable better use of healthcare-related data. However, critical in assessing whether blockchain can fulfill the hype of a technology characterized as 'revolutionary' and 'disruptive', is the need to ensure that blockchain design elements consider actual healthcare needs from the diverse perspectives of consumers, patients, providers, and regulators. In addition, answering the real needs of healthcare stakeholders, blockchain approaches must also be responsive to the unique challenges faced in healthcare compared to other sectors of the economy. In this sense, ensuring that a health blockchain is 'fit-for-purpose' is pivotal. This concept forms the basis for this article, where we share views from a multidisciplinary group of practitioners at the forefront of blockchain conceptualization, development, and deployment.

Tecnologia Biomédica , Redes de Comunicação de Computadores , Assistência à Saúde/tendências , Sistemas de Informação Administrativa , Informática Médica , Tecnologia Biomédica/métodos , Tecnologia Biomédica/organização & administração , Tecnologia Biomédica/tendências , Redes de Comunicação de Computadores/organização & administração , Redes de Comunicação de Computadores/normas , Redes de Comunicação de Computadores/provisão & distribução , Redes de Comunicação de Computadores/tendências , Data Warehousing/métodos , Data Warehousing/tendências , Assistência à Saúde/métodos , Assistência à Saúde/organização & administração , Processamento Eletrônico de Dados/métodos , Processamento Eletrônico de Dados/organização & administração , Processamento Eletrônico de Dados/tendências , Utilização de Equipamentos e Suprimentos/organização & administração , Utilização de Equipamentos e Suprimentos/tendências , Ensaios de Triagem em Larga Escala/normas , Humanos , Sistemas de Informação Administrativa/normas , Sistemas de Informação Administrativa/tendências , Informática Médica/métodos , Informática Médica/organização & administração , Informática Médica/tendências , Registros Médicos/normas
Nat Protoc ; 12(1): 88-103, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27929521


Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Humano/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Linhagem Celular , Doxiciclina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos
Science ; 350(6264): 1101-4, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26456528


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.

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
Nat Commun ; 5: 5507, 2014 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-25425480


CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5-8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

Proteínas de Bactérias/metabolismo , Endonucleases/metabolismo , Marcação de Genes , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Polimorfismo de Nucleotídeo Único , Streptococcus pyogenes/enzimologia , Proteínas de Bactérias/química , Sequência de Bases , Endonucleases/química , Humanos , Dados de Sequência Molecular , Taxa de Mutação , Análise de Sequência de DNA , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo