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1.
Cell Rep ; 30(2): 583-597.e6, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31940498

RESUMO

Maintenance of protein homeostasis, through inducible expression of molecular chaperones, is essential for cell survival under protein-damaging conditions. The expression and DNA-binding activity of heat shock factor 2 (HSF2), a member of the heat shock transcription factor family, increase upon exposure to prolonged proteotoxicity. Nevertheless, the specific roles of HSF2 and the global HSF2-dependent gene expression profile during sustained stress have remained unknown. Here, we found that HSF2 is critical for cell survival during prolonged proteotoxicity. Strikingly, our RNA sequencing (RNA-seq) analyses revealed that impaired viability of HSF2-deficient cells is not caused by inadequate induction of molecular chaperones but is due to marked downregulation of cadherin superfamily genes. We demonstrate that HSF2-dependent maintenance of cadherin-mediated cell-cell adhesion is required for protection against stress induced by proteasome inhibition. This study identifies HSF2 as a key regulator of cadherin superfamily genes and defines cell-cell adhesion as a determinant of proteotoxic stress resistance.

2.
PLoS Genet ; 15(10): e1008355, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584931

RESUMO

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Núcleo Celular/genética , Cromátides/genética , Dano ao DNA/genética , Genoma Humano/genética , Células HEK293 , Humanos , Mutação
3.
PLoS Genet ; 15(8): e1008013, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437150

RESUMO

Teleost fishes, thanks to their rapid evolution of sex determination mechanisms, provide remarkable opportunities to study the formation of sex chromosomes and the mechanisms driving the birth of new master sex determining (MSD) genes. However, the evolutionary interplay between the sex chromosomes and the MSD genes they harbor is rather unexplored. We characterized a male-specific duplicate of the anti-Müllerian hormone (amh) as the MSD gene in Northern Pike (Esox lucius), using genomic and expression evidence as well as by loss-of-function and gain-of-function experiments. Using RAD-Sequencing from a family panel, we identified Linkage Group (LG) 24 as the sex chromosome and positioned the sex locus in its sub-telomeric region. Furthermore, we demonstrated that this MSD originated from an ancient duplication of the autosomal amh gene, which was subsequently translocated to LG24. Using sex-specific pooled genome sequencing and a new male genome sequence assembled using Nanopore long reads, we also characterized the differentiation of the X and Y chromosomes, revealing a small male-specific insertion containing the MSD gene and a limited region with reduced recombination. Our study reveals an unexpectedly low level of differentiation between a pair of sex chromosomes harboring an old MSD gene in a wild teleost fish population, and highlights both the pivotal role of genes from the amh pathway in sex determination, as well as the importance of gene duplication as a mechanism driving the turnover of sex chromosomes in this clade.


Assuntos
Hormônio Antimülleriano/genética , Esocidae/fisiologia , Cromossomos Sexuais/genética , Processos de Determinação Sexual/genética , Animais , Animais Geneticamente Modificados , Mapeamento Cromossômico , Feminino , Duplicação Gênica , Técnicas de Silenciamento de Genes , Masculino , Filogenia , Sintenia
4.
Eur J Pharmacol ; 854: 398-405, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31039344

RESUMO

Hemoglobinopathies, such as ß-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of ß-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous ß-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of ß-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating ß-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.


Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Hemoglobina Fetal/metabolismo , Deleção de Genes , Terapia Genética/métodos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Talassemia beta/terapia , Sequência de Bases , Edição de Genes , Humanos , Células K562 , Proteínas Repressoras , Talassemia beta/genética , Talassemia beta/metabolismo , gama-Globinas/genética
5.
Nat Commun ; 10(1): 1288, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894545

RESUMO

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.


Assuntos
Cromatina/química , Síndrome de Cockayne/genética , Histona Acetiltransferases/genética , Histonas/metabolismo , Subunidades Proteicas/genética , Fator de Transcrição TFIIH/genética , Xeroderma Pigmentoso/genética , Acetilação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cromatina/metabolismo , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Histonas/genética , Humanos , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Cultura Primária de Células , Subunidades Proteicas/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição Genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia
6.
PLoS One ; 14(3): e0213376, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30845180

RESUMO

The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.


Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Mitocondrial/genética , Fibroblastos/fisiologia , Dosagem de Genes/genética , Mitocôndrias/genética , Modelos Animais , RNA Guia/genética
7.
PLoS Genet ; 14(8): e1007581, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30080860

RESUMO

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.


Assuntos
Proteína 2 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica no Desenvolvimento , Rombencéfalo/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sistemas CRISPR-Cas , Cromatina/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/fisiologia , Elementos Facilitadores Genéticos , Evolução Molecular , Loci Gênicos , Morfogênese/genética , Ativação Transcricional , Peixe-Zebra/embriologia
8.
Sci Rep ; 8(1): 11734, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082705

RESUMO

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.


Assuntos
Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/metabolismo , Animais , Feminino , Técnicas de Inativação de Genes/métodos , Hidrozoários/genética , Hidrozoários/metabolismo , Masculino , Mosaicismo , Ribonucleoproteínas/genética
9.
Nucleic Acids Res ; 46(W1): W242-W245, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29762716

RESUMO

CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , RNA/química , Software , Sequência de Bases , Proteína 9 Associada à CRISPR , Técnicas de Inativação de Genes , Genoma , Internet , Mutagênese , Oligonucleotídeos , Fluxo de Trabalho
10.
Stem Cells ; 36(9): 1421-1429, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29808941

RESUMO

Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.


Assuntos
Aniridia/genética , Sistemas CRISPR-Cas/fisiologia , Epitélio Anterior/metabolismo , Edição de Genes/métodos , Fator de Transcrição PAX6/genética , Aniridia/patologia , Humanos
11.
Nat Protoc ; 13(5): 946-986, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29651054

RESUMO

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Biologia Computacional/métodos , Endonucleases/metabolismo , Edição de Genes/métodos , RNA Guia/genética
12.
Transplantation ; 102(8): 1271-1278, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29688994

RESUMO

BACKGROUND: Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease, and these disease models would greatly benefit from immunodeficient rats to test different immunogenic treatments. METHODS: We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. RESULTS: As compared with Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient because they partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype because they displayed undetectable levels of T, B, and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1- or Il2rg-deficient rats and undetectable in Rats Rag1 and Il2rg (RRG) animals. Rag1- or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors, whereas animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin, and hepatocytes. Immune humanization of RRG animals was unsuccessful. CONCLUSIONS: Thus, immunodeficient RRG animals are useful recipients for long-term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.


Assuntos
Deleção de Genes , Proteínas de Homeodomínio/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Modelos Animais de Doenças , Éxons , Feminino , Genótipo , Hepatócitos/citologia , Humanos , Sistema Imunitário , Fígado/imunologia , Masculino , Mutação , Ratos , Ratos Sprague-Dawley , Transplante de Pele , Transplante Heterólogo , Transplantes
13.
Neuron ; 97(5): 1049-1062.e6, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29429939

RESUMO

A conserved organizational and functional principle of neural networks is the segregation of axon-dendritic synaptic connections into laminae. Here we report that targeting of synaptic laminae by retinal ganglion cell (RGC) arbors in the vertebrate visual system is regulated by a signaling system relying on target-derived Reelin and VLDLR/Dab1a on the projecting neurons. Furthermore, we find that Reelin is distributed as a gradient on the target tissue and stabilized by heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM). Through genetic manipulations, we show that this Reelin gradient is important for laminar targeting and that it is attractive for RGC axons. Finally, we suggest a comprehensive model of synaptic lamina formation in which attractive Reelin counter-balances repulsive Slit1, thereby guiding RGC axons toward single synaptic laminae. We establish a mechanism that may represent a general principle for neural network assembly in vertebrate species and across different brain areas.


Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Células Ganglionares da Retina/metabolismo , Serina Endopeptidases/biossíntese , Sinapses/metabolismo , Vias Visuais/metabolismo , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular Neuronais/análise , Proteínas da Matriz Extracelular/análise , Rede Nervosa/química , Proteínas do Tecido Nervoso/análise , Células Ganglionares da Retina/química , Serina Endopeptidases/análise , Sinapses/química , Vias Visuais/química , Peixe-Zebra
14.
Sci Rep ; 7(1): 16554, 2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29185448

RESUMO

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Zigoto/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Eletroporação/métodos , Feminino , Genótipo , Microscopia Confocal , Mutação , Ratos
16.
Diabetes ; 66(4): 987-993, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28082457

RESUMO

Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc-/- mice exhibit glycemic dysregulations and pancreatic ß-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.


Assuntos
Galactose/genética , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ácidos Neuramínicos/metabolismo , Antagonistas de Receptores Purinérgicos P1/farmacologia , Teofilina/farmacologia , Animais , Antígenos Heterófilos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Peptídeo C/efeitos dos fármacos , Peptídeo C/metabolismo , Diabetes Mellitus Tipo 1/cirurgia , Galactose/imunologia , Técnicas de Inativação de Genes , Glucagon/efeitos dos fármacos , Glucagon/metabolismo , Homeostase , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas , Masculino , Ácidos Neuramínicos/imunologia , Pâncreas/metabolismo , Suínos , Transplante Heterólogo
17.
Nucleic Acids Res ; 45(7): 4158-4173, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28003477

RESUMO

Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.


Assuntos
Processamento Alternativo , Proteínas Argonauta/metabolismo , HIV-1/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Sítios de Ligação , RNA Helicases DEAD-box/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Genoma Viral , Células HEK293 , HIV-1/fisiologia , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Células Jurkat , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Viral/química , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Vírion/fisiologia
18.
Mar Genomics ; 30: 55-65, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27742404

RESUMO

More and more genomes are sequenced and a great range of biological questions can be examined at the genomic level in a growing number of organisms. Testing the function of genome features, from gene networks, genome organization, conserved non-coding sequences to microRNAs, and, more generally, experimentally addressing the genotype-phenotype relationship is now possible owing to the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 revolution of genome editing. In the present review, we give a brief overview of the CRISPR/Cas9 toolbox and different strategies for genome editing currently available. We list the first examples of applications to marine organisms and also draw from studies in more common laboratory models to suggest both guidelines for design of genome editing experiments as well as discuss challenges specific to marine organisms. In addition, we discuss future perspectives, including applications of CRISPR/Cas9 to base editing and targeted reprogramming of gene transcription.


Assuntos
Organismos Aquáticos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma , Genômica/métodos , Animais , Diatomáceas/genética , Invertebrados/genética , Vertebrados/genética
19.
Genome Biol ; 17(1): 148, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27380939

RESUMO

BACKGROUND: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. RESULTS: We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. CONCLUSIONS: To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , RNA Guia/genética , Software , Algoritmos , Genoma , Internet , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética
20.
Sci Rep ; 6: 29620, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27403935

RESUMO

Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species. Here we demonstrate that the CRISPR/Cas9 system is highly efficient for genome editing in a non-model crop pest Lepidoptera, the noctuid moth Spodoptera littoralis. We knocked-out the olfactory receptor co-receptor Orco gene to investigate its function in Lepidoptera olfaction. We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation. CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals. Our work genetically defines Orco as an essential OR partner for both host and mate detection in Lepidoptera, and demonstrates that CRISPR/Cas9 is a simple and highly efficient genome editing technique in noctuid pests opening new routes for gene function analysis and the development of novel pest control strategies.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Transtornos do Olfato/genética , Spodoptera/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Insetos/genética , Mutação , Receptores Odorantes/genética , Atrativos Sexuais/genética , Spodoptera/fisiologia
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