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1.
Rinsho Ketsueki ; 60(9): 1033-1045, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597825

RESUMO

As genome editing techniques are developed, they have been applied to somatic cells in clinical use. Regarding the adaptation of these techniques in human zygotes, advances in single-cell analysis technology are expected to enhance knowledge at the molecular level, even in human preimplantation embryo development, and the application of genome editing technology for molecular functional analysis is expected. However, the use of genetic modification techniques on germlines, including zygotes, spreads to all cells of an individual and is subsequently passed down through generations. Here we explore the possibilities and challenges of applying genome editing techniques to better understand the scientific aspects of the human germline.


Assuntos
Pesquisas com Embriões/ética , Edição de Genes/ética , Edição de Genes/métodos , Desenvolvimento Embrionário , Humanos
4.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
10.
Am J Bioeth ; 19(7): 16-18, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31543067
12.
Am J Bioeth ; 19(7): 28-30, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31543069
13.
Nat Biotechnol ; 37(9): 1041-1048, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477922

RESUMO

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1-4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Clonagem Molecular , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Células HEK293 , Humanos , Petromyzon , Conformação Proteica , RNA , RNA Guia/genética , Streptococcus pyogenes , Transcriptoma
14.
Nat Biotechnol ; 37(10): 1145-1148, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548727

RESUMO

Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5-7) and in a confined TC*N sequence context. Adenine base editor-induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.


Assuntos
Aminoidrolases/metabolismo , Proteína 9 Associada à CRISPR , Desoxirribonuclease I/metabolismo , Edição de Genes/métodos , Linhagem Celular , Humanos
15.
Yi Chuan ; 41(9): 777-800, 2019 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-31549678

RESUMO

Base editing is a newly developed precise genome editing technique based on the CRISPR/Cas system. According to different base modification enzymes, the current base editing systems can be divided into cytosine base editors (CBE) and adenine base editors (ABE). They use cytosine deaminases or artificially evolved adenine deaminases to perform single-base editing, and achieve C to T (G to A) or A to G (T to C) substitutions, respectively. Due to high efficiency, independence of DNA double-strand breaks, and no need for donor DNA, base editing systems have been successfully applied in diverse species including animals, plants and other organisms since the first report in 2016. Therefore, base editing systems will have a high prospect of providing important support for gene therapy and crop genetic improvement in the future. In this review, we describe the development and current applications of base editing systems for basic research and biotechnology, highlight the challenges, and discuss the directions for future research in this important field. The information presented may facilitate interested researchers to grasp the principles of base editing, to use relevant base editing tools in their own studies, or to innovate new versions of base editing in the future.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Edição de Genes , Adenina , Aminoidrolases , Animais , Biotecnologia/tendências , Citosina , Citosina Desaminase , Plantas
16.
Yi Chuan ; 41(9): 827-835, 2019 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-31549681

RESUMO

Crop improvement by domestication and traditional breeding often results in fitness penalties and loss of genetic diversity, which greatly threatens crop production and food security under the challenging global climate. De novo domestication has been proposed as a novel strategy for crop breeding. By combining multi-omics, genome editing and synthetic biology approaches, domestication of wild or semi-wild plant species can be accelerated by rapidly introducing desirable traits without causing an associated drag on their inherent traits. In this review, we summarize the history of crop domestication, emphasize the urgency for breeding strategy innovation, and discuss recent progress of de novo crop domestication.


Assuntos
Produtos Agrícolas , Domesticação , Melhoramento Vegetal , Edição de Genes
17.
N Engl J Med ; 381(13): 1240-1247, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31509667

RESUMO

The safety of CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing-related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.).


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Infecções por HIV/terapia , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Receptores CCR5/genética , Adulto , Antirretrovirais/uso terapêutico , Contagem de Células Sanguíneas , Contagem de Linfócito CD4 , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Carga Viral
20.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1401-1410, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441611

RESUMO

Gene editing is a technique for modifying gene fragments. The novel gene editing technology focuses on the field of artificial nuclease cleavage technology, mainly ZFN technology, TALEN technology, CRISPR technology and base editing technology. The continuous improvement of gene editing technology has promoted the rapid development of agriculture, animal husbandry and biomedicine, but at the same time, technical defects and ethical controversy have brought enormous challenges to its own development. This article will briefly discuss the development and challenges of gene editing technology, as well as the views at home and abroad, and hope to inspire readers to recognize gene editing technology.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Agricultura , Animais , Endonucleases
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