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2.
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
3.
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
4.
Int J Nanomedicine ; 14: 4353-4366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354265

RESUMO

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Colesterol/química , Edição de Genes , Nanopartículas/química , Transfecção/métodos , Cátions/química , Morte Celular , Colesterol/análogos & derivados , Ensaio de Desvio de Mobilidade Eletroforética , Endossomos/metabolismo , Ácidos Graxos Monoinsaturados/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Tamanho da Partícula , Fosfatidiletanolaminas/química , Plasmídeos/metabolismo , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , RNA Guia/metabolismo , Eletricidade Estática
5.
Nat Commun ; 10(1): 3049, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296872

RESUMO

The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.


Assuntos
Ectoderma/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Placa Neural/embriologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Regulação para Baixo , Ectoderma/metabolismo , Embrião não Mamífero , Elementos Facilitadores Genéticos/genética , Epiderme/embriologia , Epiderme/metabolismo , Técnicas de Inativação de Genes , Modelos Animais , Placa Neural/metabolismo , Fosfoproteínas/genética , Ligação Proteica/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transativadores/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
6.
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
7.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
8.
Nat Commun ; 10(1): 2723, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222014

RESUMO

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Transativadores/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Epigênese Genética/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Análise de Célula Única , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
9.
BMC Bioinformatics ; 20(1): 332, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31195957

RESUMO

BACKGROUND: CRISPR-Cpf1 has recently been reported as another RNA-guided endonuclease of class 2 CRISPR-Cas system, which expands the molecular biology toolkit for genome editing. However, most of the online tools and applications to date have been developed primarily for the Cas9. There are a limited number of tools available for the Cpf1. RESULTS: We present DeepCpf1, a deep convolution neural networks (CNN) approach to predict Cpf1 guide RNAs on-target activity and off-target effects using their matched and mismatched DNA sequences. Trained on published data sets, DeepCpf1 is superior to other machine learning algorithms and reliably predicts the most efficient and less off-target effects guide RNAs for a given gene. Combined with a permutation importance analysis, the key features of guide RNA sequences are identified, which determine the activity and specificity of genome editing. CONCLUSIONS: DeepCpf1 can significantly improve the accuracy of Cpf1-based genome editing and facilitates the generation of optimized guide RNAs libraries.


Assuntos
Sistemas CRISPR-Cas/genética , Aprendizado Profundo , Endonucleases/metabolismo , Redes Neurais (Computação) , Algoritmos , Sequência de Bases , RNA Guia/genética
10.
J Vet Sci ; 20(3): e23, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31161741

RESUMO

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Ácidos Nucleicos Heteroduplexes/genética , RNA Guia/genética , Animais , Pareamento Incorreto de Bases/genética , Linhagem Celular , Edição de Genes/normas , Genes erbB-1/genética , Ácidos Nucleicos Heteroduplexes/química , RNA Guia/química , Suínos
12.
Nat Commun ; 10(1): 2603, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197149

RESUMO

During thymic negative selection, autoreactive thymocytes carrying T cell receptor (TCR) with overtly strong affinity to self-MHC/self-peptide are removed by Bim-dependent apoptosis, but how Bim is specifically regulated to link TCR activation and apoptosis induction is unclear. Here we identify a murine T cell-specific genomic enhancer EBAB (Bub1-Acoxl-Bim), whose deletion leads to accumulation of thymocytes expressing high affinity TCRs. Consistently, EBAB knockout mice have defective negative selection and fail to delete autoreactive thymocytes in various settings, with this defect accompanied by reduced Bim expression and apoptosis induction. By contrast, EBAB is dispensable for maintaining peripheral T cell homeostasis via Bim-dependent pathways. Our data thus implicate EBAB as an important, developmental stage-specific regulator of Bim expression and apoptosis induction to enforce thymic negative selection and suppress autoimmunity. Our study unravels a part of genomic enhancer codes that underlie complex and context-dependent gene regulation in TCR signaling.


Assuntos
Autoimunidade/genética , Proteína 11 Semelhante a Bcl-2/genética , Elementos Facilitadores Genéticos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Timócitos/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Autoimunidade/imunologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Sistemas CRISPR-Cas/genética , Elementos Facilitadores Genéticos/genética , Feminino , Regulação da Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Timo/citologia , Timo/imunologia
13.
Nat Commun ; 10(1): 2615, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197154

RESUMO

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing ß-carotene and co-producing ß-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.


Assuntos
Vias Biossintéticas/genética , Engenharia Metabólica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Flavanonas/biossíntese , Recombinação Homóloga/genética , Norisoprenoides/biossíntese , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Transcrição/genética , beta Caroteno/biossíntese
14.
Mol Biol (Mosk) ; 53(2): 179-199, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099770

RESUMO

At present, pharmacologically significant proteins are synthesized in different expression systems, from bacterial to mammalian and insect cell cultures. The plant expression systems (especially suspension cell culture) combine the simplicity and low cost of bacterial systems with the ability to perform eukaryotic-type posttranslational protein modifications. A low (compared with bacterial systems) yield of the target recombinant protein is one of the shortcomings of the plant expression systems. In this review, methods, developed over the past two decades, to increase the level of recombinant gene expression and methods to prevent silencing, caused by a random insertion of the target gene into heterochromatin region, are considered. The emergence of CRISPR/Cas technologies led to the creation of a new approach to increase the gene expression level, directional insertion of "pharmaceutical" protein genes in specific, knowingly transcriptionally active genome regions. The plant cell housekeeping gene loci, actively expressed throughout the interphase, are these regions. The organization of some housekeeping genes, most promising for transferring recombinant protein genes in their loci, is considered in detail.


Assuntos
Edição de Genes/tendências , Células Vegetais/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Sistemas CRISPR-Cas/genética , Genes Essenciais/genética
15.
Mol Biol (Mosk) ; 53(2): 311-323, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099781

RESUMO

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Vírus da Hepatite B , Sistemas CRISPR-Cas/genética , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/genética
16.
Nat Biotechnol ; 37(6): 626-631, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110355

RESUMO

Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4-5 nucleotides to up to ~8-9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Adenina/química , Citosina/química , Humanos , Nucleotídeos/química , Nucleotídeos/genética , Plasmídeos/química , Plasmídeos/genética
17.
Dev Growth Differ ; 61(4): 265-275, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037730

RESUMO

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Fatores de Tempo
18.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071912

RESUMO

Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.


Assuntos
Carcinoma Epitelial do Ovário/genética , Proliferação de Células/genética , N-Acetilgalactosaminiltransferases/genética , Animais , Sistemas CRISPR-Cas/genética , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Glicosilação , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Ovário/patologia , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071955

RESUMO

Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%-20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.


Assuntos
Proteínas de Fusão bcr-abl/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Técnicas de Inativação de Genes , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mesilato de Imatinib/efeitos adversos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Panobinostat/farmacologia
20.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086078

RESUMO

Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D3 (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D3 exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D3-treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D3 anti-hepatitis C virus effect. We show that vitamin D3 activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D3. This is inferred from the findings that 25(OH)D3 could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D3. Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D3 and 25(OH)D3 was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D3 anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D3 and 25(OH)D3, being 3ß-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D3 that is mediated by 25(OH)D3 in a vitamin D receptor-independent mechanism.


Assuntos
Calcifediol/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Receptores de Calcitriol/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colecalciferol/farmacologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Calcitriol/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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