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1.
Nat Methods ; 13(2): 127-37, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26820547

RESUMEN

Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators and epigenome-modifying factors to any genomic site, leading to new insights into the function of epigenetic marks in gene expression. Additionally, custom transcriptional and epigenetic regulation is facilitating refined control over cell function and decision making. The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.


Asunto(s)
Epigénesis Genética/fisiología , Epigenómica , Transcripción Genética/fisiología , Animales , Sistemas CRISPR-Cas , Regulación de la Expresión Génica , Ingeniería Genética/métodos
2.
Annu Rev Biomed Eng ; 19: 249-277, 2017 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-28633563

RESUMEN

The programming of new functions into mammalian cells has tremendous application in research and medicine. Continued improvements in the capacity to sequence and synthesize DNA have rapidly increased our understanding of mechanisms of gene function and regulation on a genome-wide scale and have expanded the set of genetic components available for programming cell biology. The invention of new research tools, including targetable DNA-binding systems such as CRISPR/Cas9 and sensor-actuator devices that can recognize and respond to diverse chemical, mechanical, and optical inputs, has enabled precise control of complex cellular behaviors at unprecedented spatial and temporal resolution. These tools have been critical for the expansion of synthetic biology techniques from prokaryotic and lower eukaryotic hosts to mammalian systems. Recent progress in the development of genome and epigenome editing tools and in the engineering of designer cells with programmable genetic circuits is expanding approaches to prevent, diagnose, and treat disease and to establish personalized theranostic strategies for next-generation medicines. This review summarizes the development of these enabling technologies and their application to transforming mammalian synthetic biology into a distinct field in research and medicine.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Redes Reguladoras de Genes/genética , Mejoramiento Genético/métodos , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Optogenética/métodos , Biología Sintética/métodos , Animales , Humanos , Análisis de Flujos Metabólicos/métodos
3.
bioRxiv ; 2024 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-38496583

RESUMEN

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

4.
Cell Rep ; 33(9): 108460, 2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-33264623

RESUMEN

Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators. We utilize deactivated Cas9 (dCas9)-based gene activation to target 1,496 putative transcription factors (TFs) in the human genome. Using a reporter of neuronal commitment, we profile the neurogenic activity of these factors in human pluripotent stem cells (PSCs), leading to a curated set of pro-neuronal factors. Activation of pairs of TFs reveals neuronal cofactors, including E2F7, RUNX3, and LHX8, that improve conversion efficiency, subtype specificity, and maturation of neuronal cell types. Finally, using multiplexed gene regulation with orthogonal CRISPR systems, we demonstrate improved neuronal differentiation with concurrent activation and repression of target genes, underscoring the power of CRISPR-based gene regulation for programming complex cellular phenotypes.


Asunto(s)
Sistemas CRISPR-Cas/genética , Regulación de la Expresión Génica/genética , Neuronas/metabolismo , Activación Transcripcional/genética , Diferenciación Celular , Humanos
5.
Nat Biotechnol ; 37(12): 1493-1501, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31548729

RESUMEN

Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.


Asunto(s)
Sistemas CRISPR-Cas/genética , Ingeniería Genética/métodos , Transcripción Genética/genética , Escherichia coli/genética , Células HEK293 , Humanos , Listeria monocytogenes/genética , ARN Guía de Kinetoplastida/genética
6.
Curr Opin Genet Dev ; 52: 13-21, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29803990

RESUMEN

The ability to reprogram cell lineage specification through the activity of master regulatory transcription factors has transformed disease modeling, drug screening, and cell therapy for regenerative medicine. Recent advances in the engineering of synthetic transcription factors to modulate endogenous gene expression networks and chromatin states have generated a new set of tools with unique advantages to study and enhance cell reprogramming methods. Several studies have applied synthetic transcription factors in various cell reprogramming paradigms in human and murine cells. Moreover, the adaption of CRISPR-based transcription factors for high-throughput screening will enable the systematic identification of optimal factors and gene network perturbations to improve current reprogramming protocols and enable conversion to more diverse, highly specified, and mature cell types. The rapid development of next-generation technologies with more robust and versatile functionality will continue to expand the application of synthetic transcription factors for cell reprogramming.


Asunto(s)
Diferenciación Celular/genética , Reprogramación Celular/genética , Cromatina/genética , Células Madre Pluripotentes Inducidas , Animales , Linaje de la Célula/genética , Humanos , Ratones , Medicina Regenerativa/tendencias
7.
Curr Opin Biotechnol ; 52: 32-41, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29500989

RESUMEN

Developments in CRISPR/Cas9-based technologies provide a new paradigm in functional screening of the genome. Conventional screening methods have focused on high-throughput perturbations of the protein-coding genome with technologies such as RNAi. However, equivalent methods for perturbing the non-coding genome have not existed until recently. CRISPR-based screening of genomic DNA has enabled the study of both genes and non-coding gene regulatory elements. Here we review recent progress in assigning function to the non-coding genome using CRISPR-based genomic and epigenomic screens, and discuss the prospects of these technologies to transforming our understanding of genome structure and regulation.


Asunto(s)
Sistemas CRISPR-Cas/genética , ADN/genética , Genómica/métodos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Epigenómica , Análisis de la Célula Individual
8.
Nat Biotechnol ; 35(6): 561-568, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28369033

RESUMEN

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the ß-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.


Asunto(s)
Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Genoma Humano/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Elementos Reguladores de la Transcripción/genética , Análisis de Secuencia de ADN/métodos , Mapeo Cromosómico/métodos , Epigenómica/métodos , Humanos
9.
Cell Stem Cell ; 19(3): 406-14, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27524438

RESUMEN

Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.


Asunto(s)
Sistemas CRISPR-Cas/genética , Epigénesis Genética , Fibroblastos/citología , Sitios Genéticos , Neuronas/citología , Transactivadores/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Marcadores Genéticos , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Ratones Endogámicos C57BL , Neuronas/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional
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