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The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.
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Enzima Convertidora de Angiotensina 2/biosíntesis , COVID-19/metabolismo , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/genética , Células CACO-2 , Catepsina L/biosíntesis , Catepsina L/genética , Chlorocebus aethiops , Humanos , Proteínas de Neoplasias/genética , Sistema Renina-Angiotensina , SARS-CoV-2/genética , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Células VeroRESUMEN
Double-stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C-NHEJ) and alternative end joining (Alt-EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt-EJ-mediated DNA repair. Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development. The polq mutant shows the same sensitivity as wild-type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild-type. Furthermore, we show that POLQ is involved in the repair of CRISPR-Cas9-induced double-stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway. This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two-sided HR-mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding.
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Bryopsida/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Secuencia de Bases , Bleomicina/farmacología , Bryopsida/efectos de los fármacos , Bryopsida/efectos de la radiación , Cisplatino/farmacología , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/genética , Inestabilidad Genómica , Recombinación Homóloga/efectos de los fármacos , Recombinación Homóloga/efectos de la radiación , Metilmetanosulfonato/farmacología , Mutación/genética , Tasa de Mutación , Fenotipo , Rayos Ultravioleta , ADN Polimerasa thetaRESUMEN
Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.
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Proteínas Bacterianas/genética , Bryopsida/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Técnicas de Sustitución del Gen , Técnicas de Transferencia de Gen , ARN Guía de Kinetoplastida/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Endonucleasas/metabolismo , Marcación de Gen/métodos , Genoma de Planta , Plantas Modificadas Genéticamente , ARN Guía de Kinetoplastida/metabolismo , Reparación del ADN por RecombinaciónRESUMEN
The ability to address the CRISPR-Cas9 nuclease complex to any target DNA using customizable single-guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single-guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end-joining (alt-EJ)-driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology-driven repair (HDR) at the target locus but also that Cas9-induced double-strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR-mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR-induced HDR is only partially mediated by the classical homologous recombination pathway.
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Proteínas de Arabidopsis/genética , Bryopsida/enzimología , Bryopsida/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Marcación de Gen/métodos , Mutagénesis , Recombinasa Rad51/genética , Adenina/análogos & derivados , Adenina/farmacología , Reparación del ADN por Unión de Extremidades , Endonucleasas , Ingeniería Genética/métodos , Genoma de Planta , Recombinación Homóloga , Plantas Modificadas Genéticamente , Protoplastos , Recombinasa Rad51/metabolismo , Eliminación de Secuencia , Homología de Secuencia , Streptococcus pyogenes/genética , Transformación GenéticaRESUMEN
KEY MESSAGE: New tools for the precise modification of crops genes are now available for the engineering of new ideotypes. A future challenge in this emerging field of genome engineering is to develop efficient methods for allele mining. Genome engineering tools are now available in plants, including major crops, to modify in a predictable manner a given gene. These new techniques have a tremendous potential for a spectacular acceleration of the plant breeding process. Here, we discuss how genetic diversity has always been the raw material for breeders and how they have always taken advantage of the best available science to use, and when possible, increase, this genetic diversity. We will present why the advent of these new techniques gives to the breeders extremely powerful tools for crop breeding, but also why this will require the breeders and researchers to characterize the genes underlying this genetic diversity more precisely. Tackling these challenges should permit the engineering of optimized alleles assortments in an unprecedented and controlled way.
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Productos Agrícolas/genética , Ingeniería Genética/métodos , Genoma de Planta/genética , Fitomejoramiento/métodos , Productos Agrícolas/crecimiento & desarrollo , Genes de Plantas/genética , Variación Genética , Fenotipo , Plantas Modificadas Genéticamente , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Influenza A viruses (IAV) pose a constant threat to human and poultry health. Of particular interest are the infections caused by highly pathogenic avian influenza (HPAI) viruses, such as H5N1, which cause significant production issues. In response to influenza infection, cells activate immune mechanisms that lead to increased interferon (IFN) production. To investigate how alterations in the interferon signaling pathway affect the cellular response to infection in the chicken, we used CRISPR/Cas9 to generate a chicken cell line that lacks a functional the type I interferon receptor (IFNAR1). We then assessed viral infections with the WSN strain of influenza. Cells lacking a functional IFNAR1 receptor showed reduced expression of the interferon stimulated genes (ISG) such as Protein Kinase R (PKR) and Myxovirus resistance (Mx) and were more susceptible to viral infection with WSN. We further investigated the role or IFNAR1 on low pathogenicity avian influenza (LPAI) strains (H7N9) and a HPAI strain (H5N1). Intriguingly, Ifnar-/- cells appeared more resistant than WT cells when infected with HPAI virus, potentially indicating a different interaction between H5N1 and the IFN signaling pathway. Our findings support that ChIFNAR1 is a key component of the chicken IFN signaling pathway and these data add contributions to the field of host-avian pathogen interaction and innate immunity in chickens.
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CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.
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Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Genoma de Planta/genética , Plantas/genética , Plantas Modificadas Genéticamente/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host-pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design and speed of vaccine supply is the most critical factor in protecting the population, as vaccination is the only sustainable defense. Vaccines need to be safe, efficient, and cost-effective, which is influenced by our understanding of the host-pathogen interface. Additionally, some of the major challenges of vaccines are the establishment of a long-lasting immunity offering cross protection to emerging strains. Although many NSVs are controlled through immunisations, for some, vaccine design has failed or efficacy has proven unreliable. The key behind designing a successful vaccine is understanding the host-pathogen interaction and the host immune response towards NSVs. In this paper, we review the recent research in vaccine design against NSVs and explore the immune responses induced by these viruses. The generation of a robust and integrated approach to development capability and vaccine manufacture can collaboratively support the management of outbreaking NSV disease health risks.
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Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1(T) and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.