RESUMEN
Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.
Asunto(s)
Sistemas CRISPR-Cas , Resistencia a Antineoplásicos , Genoma Humano , ARN Largo no Codificante/genética , Animales , Citarabina/farmacología , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Farmacogenética , Proteínas/genética , ARN/análisis , ARN Mensajero/genética , Transducción de SeñalRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins offer a breakthrough platform for cheap, programmable, and effective sequence-specific DNA targeting. The CRISPR-Cas system is naturally equipped for targeted DNA cutting through its native nuclease activity. As such, groups researching a broad spectrum of biological organisms have quickly adopted the technology with groundbreaking applications to genomic sequence editing in over 20 different species. However, the biological code of life is not only encoded in genetics but also in epigenetics as well. While genetic sequence editing is a powerful ability, we must also be able to edit and regulate transcriptional and epigenetic code. Taking inspiration from work on earlier sequence-specific targeting technologies such as zinc fingers (ZFs) and transcription activator-like effectors (TALEs), researchers quickly expanded the CRISPR-Cas toolbox to include transcriptional activation, repression, and epigenetic modification. In this review, we highlight advances that extend the CRISPR-Cas toolkit for transcriptional and epigenetic regulation, as well as best practice guidelines for these tools, and a perspective on future applications.
Asunto(s)
Sistemas CRISPR-Cas , Animales , Diferenciación Celular/genética , Epigénesis Genética , Represión Epigenética , Edición Génica , Humanos , Fenotipo , Interferencia de ARN , ARN Guía de Kinetoplastida/genética , ARN Largo no Codificante/genética , Activación TranscripcionalRESUMEN
Many microbial ecology experiments use sequencing data to measure a community's response to an experimental treatment. In a common experimental design, two units, one control and one experimental, are sampled before and after the treatment is applied to the experimental unit. The four resulting samples contain information about the dynamics of organisms that respond to the treatment, but there are no analytical methods designed to extract exactly this type of information from this configuration of samples. Here we present an analytical method specifically designed to visualize and generate hypotheses about microbial community dynamics in experiments that have paired samples and few or no replicates. The method is based on the Poisson lognormal distribution, long studied in macroecology, which we found accurately models the abundance distribution of taxa counts from 16S rRNA surveys. To demonstrate the method's validity and potential, we analyzed an experiment that measured the effect of crude oil on ocean microbial communities in microcosm. Our method identified known oil degraders as well as two clades, Maricurvus and Rhodobacteraceae, that responded to amendment with oil but do not include known oil degraders. Our approach is sensitive to organisms that increased in abundance only in the experimental unit but less sensitive to organisms that increased in both control and experimental units, thus mitigating the role of "bottle effects".
Asunto(s)
Ecología , BiodiversidadRESUMEN
We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.
Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/análisis , Sitios de Unión , Proteínas Asociadas a CRISPR/genética , Citometría de Flujo , Colorantes Fluorescentes/análisis , Eliminación de Gen , Genes Reporteros , Ingeniería Genética/métodos , Vectores Genéticos , Genoma , Células HEK293 , Humanos , Microscopía Fluorescente , Mutagénesis , Mutación , Edición de ARN , Transcripción GenéticaRESUMEN
The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
