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Background: Although imaging techniques provide information about the morphology and stability of carotid plaque, they are operator dependent and may miss certain subtleties. A variety of radiomics models for carotid plaque have recently been proposed for identifying vulnerable plaques and predicting cardiovascular and cerebrovascular diseases. The purpose of this review was to assess the risk of bias, reporting, and methodological quality of radiomics models for carotid atherosclerosis plaques. Methods: A systematic search was carried out to identify available literature published in PubMed, Web of Science, and the Cochrane Library up to March 2023. Studies that developed and/or validated machine learning models based on radiomics data to identify and/or predict unfavorable cerebral and cardiovascular events in carotid plaque were included. The basic information of each piece of included literature was identified, and the reporting quality, risk of bias, and radiomics methodology quality were assessed according the TRIPOD (Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis) checklist, the Prediction Model Risk of Bias Assessment Tool (PROBAST), and the radiomics quality score (RQS), respectively. Results: A total of 2,738 patients from 19 studies were included. The mean overall TRIPOD adherence rate was 66.1% (standard deviation 12.8%), with a range of 45-87%. All studies had a high overall risk of bias, with the analysis domain being the most common source of bias. The mean RQS was 9.89 (standard deviation 5.70), accounting for 27.4% of the possible maximum value of 36. The mean area under the curve for diagnostic or predictive properties of these included radiomics models was 0.876±0.09, with a range of 0.741-0.989. Conclusions: Radiomics models may have value in the assessment of carotid plaque, the overall scientific validity and reporting quality of current carotid plaque radiomics reports are still lacking, and many barriers must be overcome before these models can be applied in clinical practice.
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Imaging modalities provide information on plaque morphology and vulnerability; however, they are operator dependent and miss a great deal of microscopic information. Recently, many radiomics models for carotid plaque that identify unstable plaques and predict cardiovascular outcomes have been proposed. This systematic review was aimed at assessing whether radiomics is a reliable and reproducible method for the clinical prediction of carotid plaque. A systematic search was conducted to identify studies published in PubMed and Cochrane library from January 1, 2001, to September 30, 2022. Both retrospective and prospective studies that developed and/or validated machine learning models based on radiomics data to classify or predict carotid plaques were included. The general characteristics of each included study were selected, and the methodological quality of radiomics reports and risk of bias were evaluated using the radiomics quality score (RQS) tool and Quality Assessment of Diagnostic Accuracy Studies-2, respectively. Two investigators independently reviewed each study, and the consensus data were used for analysis. A total of 2429 patients from 16 studies were included. The mean area under the curve of radiomics models for diagnostic or predictive performance of the included studies was 0.88 ± 0.02, with a range of 0.741-0.989. The mean RQS was 9.25 (standard deviation: 6.04), representing 25.7% of the possible maximum value of 36, whereas the lowest point was -2, and the highest score was 22. Radiomics models have revealed additional information on patients with carotid plaque, but with respect to methodological quality, radiomics reports are still in their infancy, and many hurdles need to be overcome.
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Aprendizaje Automático , Humanos , Estudios Prospectivos , Estudios Retrospectivos , ConsensoRESUMEN
Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Drosophila , Animales , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Drosophila/genética , Exones/genética , Recombinación Homóloga , PlásmidosRESUMEN
Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.
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Proteínas de Drosophila , Animales , Proteínas Cromosómicas no Histona/genética , Drosophila/genética , Proteínas de Drosophila/genética , Heterocromatina , Homeostasis , Humanos , Receptores Notch/genéticaRESUMEN
BACKGROUND: Neuroinflammation in the spinal cord following acute brachial plexus injury (BPI) remains a vital cause that leads to motor dysfunction and neuropathic pain. In this study, we aim to explore the role of long non-coding RNA JHDM1D antisense 1 (JHDM1D-AS1) in mediating BPI-induced neuroinflammation and neuronal injury. METHODS: A total brachial plexus root avulsion (tBPRA) model in adult rats and IL-1ß-treated motor neuron-like NSC-34 cells and LPS-treated microglia cell line BV2 were conducted for in vivo and in vitro experiments, respectively. The expressions of JHDM1D-AS1, miR-101-3p and DUSP1, p38, NF-κB, TNF-α, IL-1ß, and IL-6 were detected by RT-PCR and western blot seven days after tBPI. Immunohistochemistry (IHC) was used to detect neuronal apoptosis. CCK8 assay, Tunel assay and LDH kit were used for the detection of neuronal injury. The targeted relationships between JHDM1D-AS1 and miR-101-3p, miR-101-3p and DUSP1 were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay. RESULTS: We found significant downregulated expression of JHDM1D-AS1 and DUSP1 but upregulated expression of miR-101-3p in the spinal cord after tBPI. Overexpression of JHDM1D-AS1 had a prominent neuroprotective effect by suppressing neuronal apoptosis and microglial inflammation through reactivation of DUSP1. Further exploration revealed that JHDM1D-AS1 may act as a competitive endogenous RNA targeting miR-101-3p, which bound on the 3'UTR of DUSP1 mRNA. In addition, overexpression of miR-101-3p could reverse the neuroprotective effects of JHDM1D-AS1 upregulation by blocking DUSP1. CONCLUSIONS: JHDM1D-AS1 exerted neuroprotective and anti-inflammatory effects in a rat model of tBPI by regulating miR-101-3p/DUSP1 axis.
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Neuropatías del Plexo Braquial/enzimología , MicroARNs/metabolismo , Microglía/enzimología , Neuronas Motoras/enzimología , Mielitis/enzimología , ARN Largo no Codificante/metabolismo , Médula Espinal/enzimología , Animales , Apoptosis , Neuropatías del Plexo Braquial/genética , Neuropatías del Plexo Braquial/patología , Neuropatías del Plexo Braquial/fisiopatología , Línea Celular , Modelos Animales de Enfermedad , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Ratones , MicroARNs/genética , Microglía/patología , Neuronas Motoras/patología , Mielitis/genética , Mielitis/patología , Mielitis/fisiopatología , ARN Largo no Codificante/genética , Ratas , Transducción de Señal , Médula Espinal/patología , Médula Espinal/fisiopatología , Regulación hacia ArribaRESUMEN
The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.
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Drosophila melanogaster , Drosophila , Animales , Composición de Base , Sistemas CRISPR-Cas , Drosophila/genética , Drosophila melanogaster/genética , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.
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Drosophila melanogaster/genética , Ingeniería Genética/métodos , Animales , Sistemas CRISPR-Cas/genética , Expresión Génica , Interferencia de ARN , Activación TranscripcionalRESUMEN
Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, we develop a next generation transgenic RNAi system. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, we increase versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, we have generated a collection of transgenic RNAi lines for those genes that are highly homologous to human disease genes.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4αEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.
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CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.
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Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Proteínas de Drosophila , Regulación de la Expresión Génica/genética , Factores de Transcripción , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.
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Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma , Genotipo , Larva , ARN/genética , ARN/metabolismoRESUMEN
Nowadays, genome editing tools are indispensable for studying gene function in order to increase our knowledge of biochemical processes and disease mechanisms. The extensive availability of mutagenesis and transgenesis tools make Drosophila melanogaster an excellent model organism for geneticists. Early mutagenesis tools relied on chemical or physical methods, ethyl methane sulfonate (EMS) and X-rays respectively, to randomly alter DNA at a nucleotide or chromosomal level. Since the discovery of transposable elements and the availability of the complete fly genome, specific genome editing tools, such as P-elements, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have undergone rapid development. Currently, one of the leading and most effective contemporary tools is the CRISPR-cas9 system made popular because of its low cost, effectiveness, specificity and simplicity of use. This review briefly addresses the most commonly used mutagenesis and transgenesis tools in Drosophila, followed by an in-depth review of the multipurpose CRISPR-Cas9 system and its current applications.
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Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Edición Génica/métodos , Ingeniería Genética/métodos , Genoma de los Insectos/genética , Animales , Animales Modificados Genéticamente , Modelos Genéticos , MutagénesisRESUMEN
Drosophila ovary is recognized as one of the best model systems to study stem cell biology in vivo. We had previously identified an autonomous role of the histone H1 in germline stem cell (GSC) maintenance. Here, we found that histone H1 depletion in escort cells (ECs) resulted in an increase of spectrosome-containing cells (SCCs), an ovary tumor-like phenotype. Further analysis showed that the Dpp pathway is excessively activated in these SCC cells, while the expression of bam is attenuated. In the H1-depleted ECs, both transposon activity and DNA damage had increased dramatically, followed by EC apoptosis, which is consistent with the role of H1 in other somatic cells. Surprisingly, H1-depleted ECs acquired cap cell characteristics including dpp expression, and the resulting abnormal Dpp level inhibits SCC further differentiation. Most interestingly, double knockdown of H1 and dpp in ECs can reduce the number of SCCs to the normal level, indicating that the additional Dpp secreted by ECs contributes to the germline tumor. Taken together, our findings indicate that histone H1 is an important epigenetic factor in controlling EC characteristics and a key suppressor of germline tumor.
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Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Células Germinativas/patología , Histonas/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Animales , Apoptosis , Recuento de Células , Daño del ADN , Elementos Transponibles de ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Modelos Biológicos , Fenotipo , Transducción de Señal , Transcripción Genética , Regulación hacia ArribaRESUMEN
Epigenetics plays critical roles in controlling stem cell self-renewal and differentiation. Histone H1 is one of the most critical chromatin regulators, but its role in adult stem cell regulation remains unclear. Here we report that H1 is intrinsically required in the regulation of germline stem cells (GSCs) in the Drosophila ovary. The loss of H1 from GSCs causes their premature differentiation through activation of the key GSC differentiation factor bam. Interestingly, the acetylated H4 lysine 16 (H4K16ac) is selectively augmented in the H1-depleted GSCs. Furthermore, overexpression of mof reduces H1 association on chromatin. In contrast, the knocking down of mof significantly rescues the GSC loss phenotype. Taken together, these results suggest that H1 functions intrinsically to promote GSC self-renewal by antagonizing MOF function. Since H1 and H4K16 acetylation are highly conserved from fly to human, the findings from this study might be applicable to stem cells in other systems.
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Autorrenovación de las Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Histonas/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Epigénesis Genética , Femenino , Células Germinativas/citología , Histonas/química , Histonas/genética , Masculino , Ovario/química , Ovario/metabolismoRESUMEN
Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.
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Ensamble y Desensamble de Cromatina/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Drosophila/genética , Epigénesis Genética/fisiología , Heterocromatina/fisiología , Aprendizaje/fisiología , Animales , Secuencia de Bases , Western Blotting , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina , Drosophila/fisiología , Heterocromatina/genética , Inmunoprecipitación , Datos de Secuencia Molecular , Octoxinol , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/anatomía & histología , Glándulas Salivales/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).
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Drosophila/genética , Interferencia de ARN , Acceso a la Información , Animales , Animales Modificados Genéticamente , Investigación Biomédica , Boston , Genes de Insecto , Vectores Genéticos , Facultades de MedicinaRESUMEN
Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. Little is known about the factors that determine preferential onset of fibrosis in particular tissues. Here we show that plasma membrane (PM) overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. We found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of PM is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion. Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. Our work indicates that traffic imbalances and PM topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.
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Adipocitos/fisiología , Membrana Celular/metabolismo , Colágeno/metabolismo , Drosophila/fisiología , AnimalesRESUMEN
The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.
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Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas de Drosophila/genética , Drosophila/genética , Edición Génica/métodos , Marcación de Gen/métodos , Genoma de los Insectos/genética , Animales , Edición de ARNRESUMEN
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.
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Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Drosophila melanogaster/genética , ARN Guía de Kinetoplastida/metabolismo , Alelos , Animales , Composición de Base/genética , Secuencia de Bases , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Reparación del ADN/genética , Células Germinativas/metabolismo , Patrón de Herencia/genética , Inyecciones , Datos de Secuencia Molecular , Mutagénesis/genética , Mutación/genética , Tasa de Mutación , Nucleótidos/genética , Especificidad de ÓrganosRESUMEN
Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9(D10A) nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.