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1.
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
6.
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
7.
Bioresour Technol ; 291: 121932, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31387837

RESUMO

Microalgae and cyanobacteria are easy to culture, with higher growth rates and photosynthetic efficiencies compared to terrestrial plants, and thus generating higher productivity. The concept of microalgal biorefinery is to assimilate carbon dioxide and convert it to chemical energy/value-added products, such as vitamins, carotenoids, fatty acids, proteins and nucleic acids, to be applied in bioenergy, health foods, aquaculture feed, pharmaceutical and medical fields. Therefore, microalgae are annotated as the third generation feedstock in bioenergy and biorefinery. In past decades, many studies thrived to improve the carbon sequestration efficiency as well as enhance value-added compounds from different algae, especially via genetic engineering, synthetic biology, metabolic design and regulation. From the traditional Agrobacterium-mediated transformation DNA to novel CRISPR (clustered regularly interspaced short palindromic repeats) technology applied in microalgae and cyanobacteria, this review has highlighted the genome editing technology for biorefinery that is a highly environmental friendly trend to sustainable and renewable development.


Assuntos
Cianobactérias , Microalgas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Engenharia Genética
8.
Rinsho Ketsueki ; 60(7): 810-817, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31391371

RESUMO

Acute myeloid leukemia (AML) remains a devasting disease. Progress has been made to define molecular mechanisms underlying disease pathogenesis due, in part, to the near-complete understanding of AML genome. Nonetheless, functional studies are necessary to assess the significance of AML-associated mutations and devise urgently needed therapies. Genome-wide knockout screening, employing CRISPR-Cas9 genome editing, is a powerful tool in functional genomics. In this study, genome-wide CRISPR screening was performed using mouse leukemia cell lines developed in our Center, followed by in vivo screening. Among 20,611 genes, 130 AML essential genes were identified, including clinically actionable candidates. It was shown that mRNA decapping enzyme scavenger (DCPS), an enzyme implicated in mRNA decay pathway, is essential for AML survival. ShRNA-mediated gene knockdown and DCPS inhibitor (RG3039) were employed to validate findings. RG3039 induced cell-cycle arrest and apoptosis in vitro. Furthermore, mass spectrometry analysis revealed an association between DCPS and RNA metabolic pathways, and RNA-Seq showed that RG3039 treatment induced aberrant mRNA splicing in AML cells. Importantly, RG3039 exhibited anti-leukemia effects in PDX models. These findings identify DCPS as a novel therapeutic target for AML, shedding new light on the nuclear RNA metabolic pathway in leukemogenesis.


Assuntos
Sistemas CRISPR-Cas , Leucemia Mieloide Aguda/genética , Processamento de RNA , Estabilidade de RNA , Animais , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Camundongos , RNA Interferente Pequeno
10.
Genome Biol ; 20(1): 153, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375138

RESUMO

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.


Assuntos
Resistência Microbiana a Medicamentos/genética , Metagenômica/métodos , Microbiota/genética , Análise de Sequência de DNA/métodos , Vírus/genética , Animais , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transferência Genética Horizontal , Genes Microbianos , Fases de Leitura Aberta , Prófagos/genética , Rúmen/microbiologia , Rúmen/virologia , Vírus/isolamento & purificação
11.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
12.
Life Sci ; 232: 116636, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295471

RESUMO

Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/tendências , Genoma , Humanos , Plantas/genética
13.
Microbiol Res ; 226: 1-9, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284938

RESUMO

Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.


Assuntos
Citrus/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Interferência de RNA , Bactérias/genética , Bactérias/patogenicidade , Sistemas CRISPR-Cas , Citrus/microbiologia , Produtos Agrícolas , Fungos/genética , Fungos/patogenicidade , Edição de Genes/métodos , Engenharia Genética , Vírus/genética , Vírus/patogenicidade
14.
Nat Commun ; 10(1): 3001, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278272

RESUMO

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , Staphylococcus epidermidis/genética , Thermus thermophilus/genética , Elongação da Transcrição Genética/imunologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/imunologia , RNA Guia/genética , RNA Guia/imunologia , RNA Guia/metabolismo , Staphylococcus epidermidis/imunologia , Thermus thermophilus/imunologia
15.
Genome Biol ; 20(1): 137, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300006

RESUMO

Systems for CRISPR-based combinatorial perturbation of two or more genes are emerging as powerful tools for uncovering genetic interactions. However, systematic identification of these relationships is complicated by sample, reagent, and biological variability. We develop a variational Bayes approach (GEMINI) that jointly analyzes all samples and reagents to identify genetic interactions in pairwise knockout screens. The improved accuracy and scalability of GEMINI enables the systematic analysis of combinatorial CRISPR knockout screens, regardless of design and dimension. GEMINI is available as an open source R package on GitHub at https://github.com/sellerslab/gemini .


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas Genéticas , Software , Teorema de Bayes , Epistasia Genética
17.
Cell Host Microbe ; 26(1): 1-2, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295418

RESUMO

How RNA-targeting CRISPR-Cas13 functions as a phage defense system has been mysterious. Recently in Nature, Meeske et al. (2019) demonstrate that Cas13 provides potent immunity to dsDNA phages without cutting their genome. By sensing phage transcripts and destroying RNA nonspecifically to arrest the cell into dormancy, Cas13 provides herd immunity.


Assuntos
Bacteriófagos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Bactérias
20.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
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