RESUMEN
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RESUMEN
Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.
Asunto(s)
Regulación de la Expresión Génica , Variación Genética , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1 , ARN sin Sentido/genética , ARN Largo no Codificante/genética , Receptores CCR5/genética , Regiones no Traducidas 3' , Alelos , Biomarcadores , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Membrana Celular/metabolismo , Genes Reporteros , Genotipo , Infecciones por VIH/metabolismo , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Grupos de Población/genética , Pronóstico , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CCR5/metabolismo , Carga ViralRESUMEN
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Asunto(s)
Sistemas CRISPR-Cas/genética , Mapeo Cromosómico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADNRESUMEN
Genomic variation in the untranslated region (UTR) has been shown to influence HLA class I expression level and associate with disease outcomes. Sequencing of the 3'UTR of common HLA-A alleles indicated the presence of two polyadenylation signals (PAS). The proximal PAS is conserved, whereas the distal PAS is disrupted within certain alleles by sequence variants. Using 3'RACE, we confirmed expression of two distinct forms of the HLA-A 3'UTR based on use of either the proximal or the distal PAS, which differ in length by 100 bp. Specific HLA-A alleles varied in the usage of the proximal versus distal PAS, with some alleles using only the proximal PAS, and others using both the proximal and distal PAS to differing degrees. We show that the short and the long 3'UTR produced similar mRNA expression levels. However, the long 3'UTR conferred lower luciferase activity as compared with the short form, indicating translation inhibition of the long 3'UTR. RNA affinity pull-down followed by mass spectrometry analysis as well as RNA coimmunoprecipitation indicated differential binding of Syncrip to the long versus short 3'UTR. Depletion of Syncrip by small interfering RNA increased surface expression of an HLA-A allotype that uses primarily the long 3'UTR, whereas an allotype expressing only the short form was unaffected. Furthermore, specific blocking of the proximal 3'UTR reduced surface expression without decreasing mRNA expression. These data demonstrate HLA-A allele-specific variation in PAS usage, which modulates their cell surface expression posttranscriptionally.
Asunto(s)
Regiones no Traducidas 3'/genética , Antígenos HLA-A/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Isoformas de Proteínas/genética , Sitios de Empalme de ARN/genética , Motivos de Unión al ARN/genética , Regulación de la Expresión Génica , Genotipo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Células Jurkat , Poliadenilación , Polimorfismo Genético , Unión Proteica , Procesamiento Postranscripcional del ARN , ARN Interferente Pequeño/genéticaRESUMEN
Variation in the 3' untranslated region (3'UTR) of the HLA-C locus determines binding of the microRNA Hsa-miR-148a, resulting in lower cell surface expression of alleles that bind miR-148a relative to those alleles that escape its binding. The HLA-C 3'UTR variant was shown to associate with HIV control, but like the vast majority of disease associations in a region dense with causal candidates, a direct effect of HLA-C expression level on HIV control was not proven. We demonstrate that a MIR148A insertion/deletion polymorphism associates with its own expression levels, affecting the extent to which HLA-C is down-regulated, the level of HIV control, and the risk of Crohn disease only among those carrying an intact miR-148a binding site in the HLA-C 3'UTR. These data illustrate a direct effect of HLA-C expression level on HIV control that cannot be attributed to other HLA loci in linkage disequilibrium with HLA-C and highlight the rich complexity of genetic interactions in human disease.
Asunto(s)
Secuencia de Bases , Enfermedad de Crohn/genética , Infecciones por VIH/genética , Antígenos HLA-C/genética , Mutación INDEL , Desequilibrio de Ligamiento , MicroARNs/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/inmunología , Alelos , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Antígenos HLA-C/biosíntesis , Antígenos HLA-C/inmunología , Humanos , Masculino , MicroARNs/biosíntesis , MicroARNs/inmunologíaRESUMEN
A recent genome-wide association study (GWAS) involving patients with hemophilia A who were exposed to but uninfected with human immunodeficiency virus type 1 (HIV-1) did not reveal genetic variants associated with resistance to HIV-1 infection, beyond homozygosity for CCR5-Δ32. Since variation in HLA class I and KIR genes is not well interrogated by standard GWAS techniques, we tested whether these 2 loci were involved in protection from HIV-1 infection in the same hemophilia cohort, using controls from the general population. Our data indicate that HLA class I alleles, presence or absence of KIR genes, and functionally relevant combinations of the HLA/KIR genotypes are not involved in resistance to parenterally transmitted HIV-1 infection.
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Resistencia a la Enfermedad , Infecciones por VIH/inmunología , Hemofilia A/complicaciones , Antígenos de Histocompatibilidad Clase I/inmunología , Receptores KIR/inmunología , Estudios de Asociación Genética , HumanosRESUMEN
Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Síndrome Respiratorio y de la Reproducción Porcina/genética , Sistemas CRISPR-Cas/genética , Resistencia a la Enfermedad/genética , Edición Génica , GanadoRESUMEN
Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.
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Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Escherichia coli , Genoma/genética , Células HEK293 , Humanos , Modelos GenéticosRESUMEN
BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N4GATT-3'), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.