RESUMEN
Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Genoma , Ácido Abscísico/farmacología , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cuerpos Enrollados/genética , Regulación de la Expresión Génica , Sitios Genéticos , Humanos , Hibridación Fluorescente in Situ , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacosRESUMEN
We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Hibridación Fluorescente in Situ/métodos , Línea Celular Tumoral , ADN/análisis , Roturas del ADN de Doble Cadena , Vectores Genéticos , Células HEK293 , Humanos , Microscopía Fluorescente , Imagen Molecular , ARN/análisis , ARN Guía de Kinetoplastida/genética , Linfocitos TRESUMEN
Nosocomial infections affect a significant number of intensive care unit (ICU) patients including those in the neurosurgical ICU. Gram-positive organisms are responsible for many of these infections and often these pathogens are resistant to some of the older antimicrobial agents. Two new classes of antibiotics have been developed: streptogramins and oxazolidinones. Linezolid is an oxazolidinone, which has been shown to be effective against methicillin- and vancomycin-resistant Gram-positive pathogens. It may be administered orally or parenterally, and displays favorable pharmacokinetic properties, with rapid and complete absorption after oral administration. Linezolid is generally well tolerated with mild gastrointestinal related adverse effects. Linezolid provides a useful alternative in the treatment of Gram-positive infections, particularly those caused by resistant organisms. It has tremendous clinical utility, especially in the ICU where infections and multi-drug resistant rates are high and treatment options become limited.