RESUMO
Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.
Assuntos
Infecções por Coronavirus , Coronavirus , Quinases Dyrk , Animais , Humanos , Camundongos , Antígenos CD13/genética , Coronavirus/classificação , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Deltacoronavirus , Vírus da Hepatite Murina/fisiologia , Suínos , Vírus da Gastroenterite Transmissível/genética , Tirosina , Replicação Viral/fisiologia , Quinases Dyrk/metabolismoRESUMO
With the exponential growth of multi-omics data, its integration and utilization have brought unprecedented opportunities for the interpretation of gene regulation mechanisms and the comprehensive analyses of biological systems. IAnimal (https://ianimal.pro/), a cross-species, multi-omics knowledgebase, was developed to improve the utilization of massive public data and simplify the integration of multi-omics information to mine the genetic mechanisms of objective traits. Currently, IAnimal provides 61 191 individual omics data of genome (WGS), transcriptome (RNA-Seq), epigenome (ChIP-Seq, ATAC-Seq) and genome annotation information for 21 species, such as mice, pigs, cattle, chickens, and macaques. The scale of its total clean data has reached 846.46 TB. To better understand the biological significance of omics information, a deep learning model for IAnimal was built based on BioBERT and AutoNER to mine 'gene' and 'trait' entities from 2 794 237 abstracts, which has practical significance for comprehending how each omics layer regulates genes to affect traits. By means of user-friendly web interfaces, flexible data application programming interfaces, and abundant functional modules, IAnimal enables users to easily query, mine, and visualize characteristics in various omics, and to infer how genes play biological roles under the influence of various omics layers.
Assuntos
Bases de Dados Genéticas , Animais , Regulação da Expressão Gênica , Genoma , Bases de Conhecimento , Software , MultiômicaRESUMO
African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Suínos , Animais , Vírus da Febre Suína Africana/genética , Sus scrofa , Linhagem Celular , RimRESUMO
Emerging coronaviruses (CoVs) pose a severe threat to human and animal health worldwide. To identify host factors required for CoV infection, we used α-CoV transmissible gastroenteritis virus (TGEV) as a model for genome-scale CRISPR knockout (KO) screening. Transmembrane protein 41B (TMEM41B) was found to be a bona fide host factor involved in infection by CoV and three additional virus families. We found that TMEM41B is critical for the internalization and early-stage replication of TGEV. Notably, our results also showed that cells lacking TMEM41B are unable to form the double-membrane vesicles necessary for TGEV replication, indicating that TMEM41B contributes to the formation of CoV replication organelles. Lastly, our data from a mouse infection model showed that the KO of this factor can strongly inhibit viral infection and delay the progression of a CoV disease. Our study revealed that targeting TMEM41B is a highly promising approach for the development of broad-spectrum anti-viral therapeutics.
Assuntos
Sistemas CRISPR-Cas , Gastroenterite Suína Transmissível/virologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/fisiologia , Organelas/virologia , Vírus da Gastroenterite Transmissível/fisiologia , Replicação Viral , Animais , Gastroenterite Suína Transmissível/genética , Gastroenterite Suína Transmissível/transmissão , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , SuínosRESUMO
As the essential tissue for sperm maturation and storage, the epididymis secretes a number of tissue-specific proteins to exert its functions. Among these proteins, epididymal lipocalins have been intensively studied because of their epididymis-specific expression pattern and clustered genomic organization. In this study, rLcn13, a member of the rat epididymal lipocalin family, is identified and elaborately characterized. The cDNA sequence of rLcn13 consists of 719 nucleotides and encodes a 176 amino-acid protein with a predicted N-terminal signal peptide of 19 amino acids. rLcn13 shares a similar genomic structure and predicted 3D protein structure with other lipocalin family members. A recombinant rLCN13 mature peptide of 157 amino acids is expressed and purified, which is used to raise a polyclonal antibody against rLCN13 with high specificity and sensitivity. Northern blot, western blot, and immunohistochemistry assays reveal that rLcn13 is an epididymis-specific gene which is expressed predominantly in the initial segment and proximal caput epididymis and influenced by androgen. The rLCN13 protein is modified by N-glycosylation and secreted into the epididymal lumen, and then binds to the acrosome region of the sperm. Our data demonstrate that rLcn13 exhibits a specific temporospatial expression pattern and androgen dependence, indicating its potential roles in sperm maturation.
Assuntos
Androgênios , Lipocalinas , Ratos , Masculino , Animais , Sequência de Aminoácidos , Lipocalinas/genética , Lipocalinas/metabolismo , Androgênios/metabolismo , Epididimo , Sêmen/metabolismo , Espermatozoides/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.
Assuntos
Calreticulina , Vírus da Encefalite Japonesa (Espécie) , Vírus da Encefalite Japonesa (Subgrupo) , Animais , Humanos , Antivirais , Calreticulina/genética , Sistemas CRISPR-Cas/genética , Citosina , Vírus da Encefalite Japonesa (Espécie)/genética , Edição de Genes , Íntrons/genética , Mamíferos , Mutação , RNA Guia de Sistemas CRISPR-Cas , SuínosRESUMO
Influenza A viruses (IAVs) are a major global health threat and in the future, may cause the next pandemic. Although studies have partly uncovered the molecular mechanism of IAV-host interaction, it requires further research. In this study, we explored the roles of transportin-3 (TNPO3) in IAV infection. We found that TNPO3-deficient cells inhibited infection with four different IAV strains, whereas restoration of TNPO3 expression in knockout (KO) cells restored IAV infection. TNPO3 overexpression in wild-type (WT) cells promoted IAV infection, suggesting that TNPO3 is involved in the IAV replication. Furthermore, we found that TNPO3 depletion restrained the uncoating in the IAV life cycle, thereby inhibiting the process of viral ribonucleoprotein (vRNP) entry into the nucleus. However, KO of TNPO3 did not affect the virus attachment, endocytosis, or endosomal acidification processes. Subsequently, we found that TNPO3 can colocalize and interact with viral proteins M1 and M2. Taken together, the depletion of TNPO3 inhibits IAV uncoating, thereby inhibiting IAV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of IAV replication and treating influenza disease.
Assuntos
Vírus da Influenza A , Influenza Humana , Humanos , Vírus da Influenza A/metabolismo , Carioferinas/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , beta Carioferinas/metabolismoRESUMO
Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.
Assuntos
Vírus da Influenza A/fisiologia , N-Acilneuraminato Citidililtransferase/antagonistas & inibidores , Infecções por Orthomyxoviridae/virologia , Receptores de Superfície Celular/metabolismo , Sialiltransferases/antagonistas & inibidores , Replicação Viral , Animais , Sistemas CRISPR-Cas , Ácido N-Acetilneuramínico/metabolismo , N-Acilneuraminato Citidililtransferase/genética , N-Acilneuraminato Citidililtransferase/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , SuínosRESUMO
Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR-Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR-Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR-Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre-clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR-Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR-Cas-modified organisms/products.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Agricultura , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Plantas/genéticaRESUMO
MOTIVATION: Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. RESULTS: The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531 306 and 2 941 669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. AVAILABILITY AND IMPLEMENTATION: sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Pequeno RNA não Traduzido/genética , Algoritmos , Primers do DNA , Reação em Cadeia da Polimerase , SoftwareRESUMO
Embryonic and neonatal skeletal muscles grow via the proliferation and fusion of myogenic cells, whereas adult skeletal muscle adapts largely by remodelling pre-existing myofibers and optimizing metabolic balance. It has been reported that miRNAs played key roles during skeletal muscle development through targeting different genes at post-transcriptional level. In this study, we show that a single miRNA (miR-208b) can modulate both the myogenesis and homoeostasis of skeletal muscle by distinct targets. As results, miR-208b accelerates the proliferation and inhibits the differentiation of myogenic cells by targeting the E-protein family member transcription factor 12 (TCF12). Also, miR-208b can stimulate fast-to-slow fibre conversion and oxidative metabolism programme through targeting folliculin interacting protein 1 (FNIP1) but not TCF12 gene. Further, miR-208b could active the AMPK/PGC-1a signalling and mitochondrial biogenesis through targeting FNIP1. Thus, miR-208b could mediate skeletal muscle development and homoeostasis through specifically targeting of TCF12 and FNIP1.
Assuntos
Metabolismo Energético , Regulação da Expressão Gênica no Desenvolvimento , Homeostase , MicroRNAs/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Interferência de RNA , Animais , Diferenciação Celular/genética , Células Cultivadas , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Mioblastos/citologia , Mioblastos/metabolismo , RNA Mensageiro/genéticaRESUMO
The epididymis is a male reproductive organ involved in posttesticular sperm maturation and storage, but the mechanism underlying sperm maturation remains unclear. ß-Defensins (Defbs) belong to a family of small, cysteine-rich, cationic peptides that are antimicrobial and modulate the immune response. A large number of Defb genes are expressed abundantly in the male reproductive tract, especially in the epididymis. We and other groups have shown the involvement of several Defb genes in regulation of sperm function. In this study, we found that Defb23, Defb26, and Defb42 were highly expressed in specific regions of the epididymis. Rats with CRISPR/Cas9-mediated single-gene disruption of Defb23, Defb26, or Defb42 had no obvious fertility phenotypes. Those with the deletion of Defb23/ 26 or Defb23/ 26/ 42 became subfertile, and sperm isolated from the epididymal cauda of multiple-mutant rats were demonstrated decreased motility. Meanwhile, the sperm showed precocious capacitation and increased spontaneous acrosome reaction. Consistent with premature capacitation and acrosome reaction, sperm from multiple-gene-knockout rats had significantly increased intracellular calcium. These results suggest that Defb family members affect sperm maturation by a synergistic pattern in the epididymis.-Zhang, C., Zhou, Y., Xie, S., Yin, Q., Tang, C., Ni, Z., Fei, J., Zhang, Y. CRISPR/Cas9-mediated genome editing reveals the synergistic effects of ß-defensin family members on sperm maturation in rat epididymis.
Assuntos
Sistemas CRISPR-Cas , Epididimo , Fertilidade , Edição de Genes , Maturação do Esperma , Motilidade dos Espermatozoides/fisiologia , beta-Defensinas/fisiologia , Animais , Técnicas de Inativação de Genes , Genoma , Masculino , Fenótipo , Ratos , beta-Defensinas/antagonistas & inibidoresRESUMO
Precise nucleic acid editing technologies have facilitated the research of cellular function and the development of novel therapeutics, especially the current programmable nucleases-based editing tools, such as the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases (Cas). As CRISPR-based therapies are advancing toward human clinical trials, it is important to understand how natural genetic variation in the human population may affect the results of these trials and even patient safety. The development of "base-editing" technique allows the direct, stable transformation of target DNA base into an alternative in a programmable way, without DNA double strand cleavage or a donor template. Genome-editing techniques hold promises for the treatment of genetic disease at the DNA level by blocking the sequences associated with disease from producing disease-causing proteins. Currently, scientists can select the gene they want to modify, use the Cas9 as a "molecular cutter" to cut it out, and transform it into a more desirable version. In this review, we focus on the recent advances of CRISPR/Cas system by outlining the evolutionary and biotechnological implications of current strategies for improving the specificity and accuracy of these genome-editing technologies.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Doenças Genéticas Inatas/terapia , Biotecnologia/tendências , Edição de Genes/tendências , Doenças Genéticas Inatas/genética , HumanosRESUMO
MicroRNAs (miRNAs) are 18-24 nucleotides non-coding RNAs that regulate gene expression by post-transcriptional suppression of mRNA. The Chinese giant salamander (CGS, Andrias davidianus), which is an endangered species, has become one of the important models of animal evolution; however, no miRNA studies on this species have been conducted. In this study, two small RNA libraries of CGS ovary and testis were constructed using deep sequencing technology. A bioinformatics pipeline was developed to distinguish miRNA sequences from other classes of small RNAs represented in the sequencing data. We found that many miRNAs and other small RNAs such as piRNA and tsRNA were abundant in CGS tissue. A total of 757 and 756 unique miRNAs were annotated as miRNA candidates in the ovary and testis respectively. We identified 145 miRNAs in CGS ovary and 155 miRNAs in CGS testis that were homologous to those in Xenopus laevis ovary and testis respectively. Forty-five miRNAs were more highly expressed in ovary than in testis and 21 miRNAs were more highly expressed in testis than in ovary. The expression profiles of the selected miRNAs (miR-451, miR-10c, miR-101, miR-202, miR-7a and miR-499) had their own different roles in other eight tissues and different development stages of testis and ovary, suggesting that these miRNAs play vital regulatory roles in sexual differentiation, gametogenesis and development in CGS. To our knowledge, this is the first study to reveal miRNA profiles that are related to male and female CGS gonads and provide insights into sex differences in miRNA expression in CGS.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Ovário/metabolismo , Testículo/metabolismo , Urodelos/genética , Animais , Biologia Computacional , Feminino , Masculino , Ovário/citologia , Testículo/citologia , Urodelos/classificaçãoRESUMO
The epididymis, which connects the testis to vas deferens, plays a crucial role regulating sperm maturation and fertilization. Here, a tamoxifen-inducible CreERT2 recombinase transgenic mouse was generated to study the function of genes in the caput epididymis using the Cre/LoxP system, which is driven by the 1.8-kb Lcn5 promoter (Lcn5-CreERT2 ). Both CRE recombinase and ERT2 mRNA were specifically expressed in the caput epididymis, beginning at postnatal Day 30 and increasing thereafter. Crossing these Lcn5-CreERT2 transgenic mice with Rosa26; mT/mG reporter mice, which express membrane-bound GFP (mGFP) only after CRE is active at its genetic locus, resulted in the presence of GFP only in the middle/distal caput epididymis after tamoxifen induction. Efficiency of the CRE recombinase production in the caput epididymis was dose- and time-dependent. These tamoxifen-inducible caput epididymis-specific CRE recombinase transgenic mice thus provides a simple approach to modulate epididymal principal cells in vivo, allowing for the genetic investigation of caput epididymis-specific gene functions during sperm maturation. 84: 257-264, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Deleção de Genes , Integrases , Proteínas Plasmáticas de Ligação ao Retinol , Tamoxifeno/farmacologia , Animais , Epididimo/metabolismo , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Plasmáticas de Ligação ao Retinol/genética , Proteínas Plasmáticas de Ligação ao Retinol/metabolismoRESUMO
The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).
Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Software , Animais , Galinhas , Biologia Computacional/métodos , Humanos , Internet , Camundongos , MicroRNAs/genética , Linguagens de Programação , Precursores de RNA/genética , Ratos , Reprodutibilidade dos Testes , Suínos , Interface Usuário-ComputadorRESUMO
The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully applied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful genome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have advantages and disadvantages respectively. In this review, we summarize characters of 16 kinds online and standalone software for sgRNA design and/or off-target evaluation and conduct a comparative analysis of these different kinds of software through developing 38 evaluation indexes. We also summarize 11 experimental approaches for testing genome editing efficiency and off-target effects as well as how to screen highly efficient and specific sgRNA.
Assuntos
Sistemas CRISPR-Cas/genética , Genoma/genética , Edição de RNA , RNA Guia de Cinetoplastídeos/genética , Humanos , SoftwareRESUMO
Appropriate innate immune responses are required to protect an organism against foreign pathogens, and the immune response must be tightly controlled. Here, we report a new microRNA (miRNA) identified from a small RNA library from the epididymis, termed miR-7578, that acts as a negative regulator of inflammatory responses. It was abundantly expressed in immune-related organs and induced by lipopolysaccharide in the lung and epididymis, as well as macrophages stimulated with diverse Toll-like receptor ligands, in an NF-κB-dependent manner. mmu-miR-7578 inhibited the release of pro-inflammatory cytokines, including TNFα and IL6, by regulating its target gene Egr1, which encodes a transcription factor that activates TNFα and NF-κB expression. Transgenic mice overexpressing mmu-miR-7578 displayed higher resistance to endotoxin shock and lower plasma levels of TNFα and IL6, indicating that this miRNA acted as a negative molecule of immune response. In sum, we report a previously uncharacterized LPS-responsive miRNA that controls inflammatory response in a feedback loop by fine-tuning a key transcription factor in vivo.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Regulação da Expressão Gênica , Interleucina-6/biossíntese , Macrófagos/metabolismo , MicroRNAs/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
MicroRNAs are involved in a number of cellular processes; thus, their deregulation is usually apt to the occurrence of diverse diseases. Previous studies indicate that abnormally up-regulated miR-29a is associated with several diseases, such as human acute myeloid leukemia and diabetes; therefore, the proper level of miR-29a is critical for homeostasis. Herein, we observed that miR-29a was repressed by androgen/androgen receptor signaling in mouse epididymis by targeting a conserved androgen response element located 8 kb upstream of miR-29b1a loci. It is well known that multiple regulatory programs often form a complicated network. Here, we found that miR-29a reversibly suppressed androgen receptor and its target genes by targeting IGF1 and p53 pathways. miR-29b1a-overexpressing transgenic mice displayed epididymis hypoplasia partially similar to the phenotype of those mice with an impaired androgen-androgen receptor signal system. Taken together, the results demonstrated that there is a regulatory circuitry between the androgen signaling pathway and miR-29a in mouse epididymis that may be vital for epididymal development and functions.
Assuntos
Epididimo/metabolismo , MicroRNAs/genética , Receptores Androgênicos/genética , Transdução de Sinais/genética , Androgênios/farmacologia , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Orquiectomia , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cell proliferation often decreases gradually during postnatal development of some organs. However, the underlying molecular mechanisms remain unclear. Epididymis, playing important roles in sperm maturation, is a typical organ of this type, which displays a decreased proliferation during postnatal development and even ceased at the adult stage. Here, epididymis was employed as a model to explore the underlying mechanisms. We profiled the microRNA and mRNA expression of newborn (1 day) and adult (90 day) rat epididymis by microarray analysis, and found that the level of miR-29a was dramatically up-regulated during postnatal development of rat epididymis. Subsequent investigations demonstrated that overexpression of miR-29a inhibited the proliferation of epididymal epithelial cells in vitro. The nuclear autoantigenic sperm protein (NASP), a novel target of miR-29a, was significantly down-regulated during postnatal development of rat epididymis. Further analysis showed that silence of NASP mimicked the anti-proliferation effect of miR-29a, whereas overexpression of this protein attenuated the effect of miR-29a. As in rat epididymis, miR-29a was up-regulated and Nasp was down-regulated during postnatal development of mouse epididymis, heart, liver, and lung. Moreover, miR-29a can also inhibit the proliferation of cancer cells by targeting Nasp. Thus, an increase of miR-29a, and hence decrease of Nasp, may contribute to inhibit cell proliferation during postnatal organ development.