RESUMEN
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
Asunto(s)
COVID-19/genética , Infecciones por Coronavirus/genética , Coronavirus/fisiología , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Células A549 , Animales , Vías Biosintéticas/efectos de los fármacos , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Colesterol/biosíntesis , Colesterol/metabolismo , Análisis por Conglomerados , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resfriado Común/genética , Resfriado Común/virología , Coronavirus/clasificación , Infecciones por Coronavirus/virología , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Ratones , Fosfatidilinositoles/biosíntesis , Células Vero , Internalización del Virus/efectos de los fármacos , Replicación ViralRESUMEN
The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.
Asunto(s)
Antibacterianos/farmacología , Sistemas CRISPR-Cas , Biología Computacional/métodos , Farmacorresistencia Bacteriana/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/genética , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/genética , Infecciones Bacterianas/prevención & control , Farmacorresistencia Bacteriana/genética , Humanos , Metagenómica/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.