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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
2.
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
3.
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
4.
BMC Plant Biol ; 19(1): 333, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370789

RESUMO

BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.


Assuntos
Edição de Genes/métodos , Genes de Plantas/genética , Gliadina/genética , Glutens/genética , Triticum/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Eletroforese em Gel de Poliacrilamida , Glutens/imunologia , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas , Alinhamento de Sequência
5.
Adv Exp Med Biol ; 1158: 269-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452146

RESUMO

Mitochondria play a central role in maintaining normal cellular homeostasis as well as contributing to the pathogenesis of numerous disease states. The advent of CRISPR-Cas9 screening technologies has greatly accelerated the study of mitochondrial biology. In this chapter, we review the various CRISPR-Cas9 screening platforms that are currently available and prior studies that leveraged this technology to identify genes involved in mitochondrial biology in both healthy and disease states. In addition, we discuss the challenges associated with current CRISPR-Cas9 platforms and potential solutions to further enhance this promising technology.


Assuntos
Sistemas CRISPR-Cas , Mitocôndrias , Doenças Mitocondriais , Fenômenos Fisiológicos Celulares/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Pesquisa/tendências
6.
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
7.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(8): 824-829, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31416510

RESUMO

OBJECTIVE: To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology. METHODS: At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice. RESULTS: The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P<0.001). CONCLUSIONS: A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.


Assuntos
Sistemas CRISPR-Cas , Erros Inatos do Metabolismo dos Aminoácidos , Animais , Proteínas de Transporte , Heterozigoto , Camundongos , Mutação
8.
Pestic Biochem Physiol ; 158: 32-39, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378358

RESUMO

Mutations in the GABA-gated chloride channel are associated with resistance to cyclodiene organochlorine and phenyl pyrazole insecticides. The best characterised of these is A301S, which was initially identified in a Dieldrin resistant strain of Drosophila melanogaster. The orthologous mutation has been found in a variety of different crop pests including the diamond back moth Plutella xylostella. However, the contribution of this mutation to resistance in this species remains unclear. We have used the CRISPR/Cas9 system in order to edit Plutella xylostella PxGABARalpha1 to Serine at the 301 orthologous position (282 in PxGABARalpha1) in an insecticide sensitive strain isolated from Vero Beach (VB) USA. In this edited line, no high level of resistance is conferred to Dieldrin, Endosulfan or Fipronil, rather only a subtle shift in sensitivity which could not confer commercially important resistance. We conclude that the high level of commercial resistance to cyclodiene organochlorine and phenyl pyrazole insecticides observed in some field isolates of Plutella xylostella cannot arise from A282S in PxGABARalpha1 alone.


Assuntos
Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Animais , Sistemas CRISPR-Cas/genética , Dieldrin/farmacologia , Endossulfano/farmacologia , Resistência a Inseticidas/genética , Mariposas/genética , Mutação/genética , Pirazóis/farmacologia , Receptores de GABA-A/genética
9.
Sheng Li Xue Bao ; 71(4): 588-596, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440756

RESUMO

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2-/Y mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2-/Y mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Camundongos Knockout , RNA Guia/genética , Animais , Feminino , Marcação de Genes , Masculino , Camundongos
10.
Genome Biol ; 20(1): 133, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287004

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells. RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA. CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.


Assuntos
Doença da Artéria Coronariana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Sistemas CRISPR-Cas , Células Endoteliais/metabolismo , Epigenômica , Estudo de Associação Genômica Ampla , Humanos , Elementos Reguladores de Transcrição , Transcriptoma
11.
Cell Mol Biol Lett ; 24: 44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31285745

RESUMO

Background: Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor ß (TGF-ß). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-ß1 during the rapid growth of sika deer antler. Methods: Three CRISPR-Cas9 knockout vectors targeting the TGF-ß1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-ß1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-ß pathway-related genes were determined using a PCR array. Results: Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-ß1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-ß signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-ß pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role. Conclusions: TGF-ß1 is a newly identified regulatory factor of rapid growth in sika deer antler.


Assuntos
Cartilagem/metabolismo , Proliferação de Células , Cervos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Animais Geneticamente Modificados , Chifres de Veado , Sistemas CRISPR-Cas , Cartilagem/fisiologia , Linhagem Celular , Cervos/genética , Cervos/fisiologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Masculino , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/fisiologia
13.
BMC Plant Biol ; 19(1): 311, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307375

RESUMO

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds. RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.


Assuntos
Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Genes de Plantas , RNA Guia , Soja/genética , Ácido alfa-Linoleico/genética , Agrobacterium/genética , Sistemas CRISPR-Cas , Marcadores Genéticos , Vetores Genéticos , Técnicas de Genotipagem , Padrões de Herança , Plantas Geneticamente Modificadas
14.
Genome Biol ; 20(1): 132, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262344

RESUMO

CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However, precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an efficient and highly practical platform for DNA detection.


Assuntos
Sistemas CRISPR-Cas , DNA/análise , Técnicas Genéticas , Testes Genéticos/métodos , Escherichia coli , Humanos , Sensibilidade e Especificidade
16.
BMC Plant Biol ; 19(1): 292, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272394

RESUMO

BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.


Assuntos
Brassicaceae/genética , Globulinas/genética , Proteínas de Plantas/genética , Proteínas de Armazenamento de Sementes/genética , Sequência de Bases , Brassicaceae/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Globulinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/genética
18.
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
19.
Yi Chuan ; 41(7): 582-598, 2019 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-31307968

RESUMO

Gene editing is a genetic manipulation technology which utilizes bacterial nucleases to accurately and efficiently modify DNA or RNA. Gene editing has broad applications in basic research, breeding, and drug screening, and it is gaining validity and applicability to the therapy of many diseases especially genetic-based disease. In this review, we summarize the development of gene editing technology, its different strategies and applications in the treatment of disease, and the research of gene editing therapy for genetic diseases (including base editor and epigenetic regulation) in the treatment of disorders and diseases of the blood system, liver, muscle and nervous system. Finally, we discuss the future development prospects of gene editing therapy.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/tendências , Terapia Genética , Doença , Epigênese Genética , Humanos
20.
Nat Commun ; 10(1): 2960, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273196

RESUMO

Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Células Clonais , Biblioteca Gênica , Engenharia Genética , Genoma Fúngico , Proteínas de Fluorescência Verde/metabolismo , Proteínas Nucleares/metabolismo
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