Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CRISPR/Cas9-Based Engineering of the Epigenome.

ABSTRACT: Determining causal relationships between distinct chromatin features and gene expression, and ultimately cell behavior, remains a major challenge. Recent developments in targetable epigenome-editing tools enable us to assign direct transcriptional and functional consequences to locus-specific chromatin modifications. This Protocol Review discusses the unprecedented opportunity that CRISPR/Cas9 technology offers for investigating and manipulating the epigenome to facilitate further understanding of stem cell biology and engineering of stem cells for therapeutic applications. We also provide technical considerations for standardization and further improvement of the CRISPR/Cas9-based tools to engineer the epigenome.

SUBMITTER: Pulecio J

PROVIDER: S-EPMC6205890 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

CRISPR/Cas9-Based Engineering of the Epigenome.

Pulecio Julian J Verma Nipun N Mejía-Ramírez Eva E Huangfu Danwei D Raya Angel A

Cell stem cell 20171001 4

Determining causal relationships between distinct chromatin features and gene expression, and ultimately cell behavior, remains a major challenge. Recent developments in targetable epigenome-editing tools enable us to assign direct transcriptional and functional consequences to locus-specific chromatin modifications. This Protocol Review discusses the unprecedented opportunity that CRISPR/Cas9 technology offers for investigating and manipulating the epigenome to facilitate further understanding ...[more]

PMID: 28985525

Similar Datasets

Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering.

Project description:CRISPR-based epigenome editing uses dCas9 as a platform to recruit transcription or chromatin regulators at chosen loci. Despite recent and ongoing advances, the full potential of these approaches to studying chromatin functions in vivo remains challenging to exploit. In this review we discuss how recent progress in plants and animals provides new routes to investigate the function of chromatin regulators and address the complexity of associated regulations that are often interconnected. While efficient transcriptional engineering methodologies have been developed and can be used as tools to alter the chromatin state of a locus, examples of direct manipulation of chromatin regulators remain scarce in plants. These reports also reveal pitfalls and limitations of epigenome engineering approaches that are nevertheless informative as they are often associated with locus- and context-dependent features, which include DNA accessibility, initial chromatin and transcriptional state or cellular dynamics. Strategies implemented in different organisms to overcome and even take advantage of these limitations are highlighted, which will further improve our ability to establish the causality and hierarchy of chromatin dynamics on genome regulation.

| S-EPMC8594717 | biostudies-literature

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.

Project description:Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

| S-EPMC4430400 | biostudies-literature

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Project description:Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

| S-EPMC4231726 | biostudies-literature

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.

Project description:CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.

| S-EPMC4877446 | biostudies-other

Chromosome engineering in zygotes with CRISPR/Cas9.

Project description:Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection.

| S-EPMC4819711 | biostudies-literature

Genome engineering using the CRISPR-Cas9 system.

Project description:Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

| S-EPMC3969860 | biostudies-literature

Improving transgene expression and CRISPR-Cas9 efficiency with molecular engineering-based molecules.

Project description:As a novel and robust gene-editing tool, the Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated protein 9 (CRISPR-Cas9) system has revolutionized gene therapy. Plasmid vector delivery is the most commonly used method for integrating the CRISPR-Cas9 system into cells. However, such foreign cytosolic DNAs trigger an innate immune response (IIR) within cells, which can hinder gene editing by inhibiting transgene expression. Although some small molecules have been shown to avoid the action of IIR on plasmids, they only work on a single target and may also affect cell viability. A genetic approach that works at a comprehensive level for manipulating IIR is still lacking. Here, we designed and constructed several artificial nucleic acid molecules (ANAMs), which are combinations of aptamers binding to two key players of IIR (β-catenin and NF-κB). ANAMs strongly inhibited the IIR in cells, thus improving transgene expression. We also used ANAMs to improve the gene-editing efficiency of the CRISPR-Cas9 system and its derivatives, thus enhancing the apoptosis of cancer cells induced by CRISPR-Cas9. ANAMs can be valuable tools for improving transgene expression and gene editing in mammalian cells.

| S-EPMC7533053 | biostudies-literature

Engineering large chromosomal deletions by CRISPR-Cas9

Project description:Arm-level chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of arm-level deletions are limited. Here we present an effective technique to engineer arm-level deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced arm-level deletions on chromosome 11q (65 MB) and chromosome 6q (53 MB) in neuroblastoma cell lines. Such isogenic models enable further research on the role of arm-level deletions in tumor development and growth, and their possible therapeutic potential.

| EGAS00001005134 | EGA

Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure.

Project description:The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones. By introducing the shRNA against DNA ligase IV gene (LIG4) into the sgRNA-shRNA construct, the HDR-based precise genome editing efficiency was improved as more than 2-fold. Our works provide a useful tool for multiplex and precise genome modifying in mammalian cells.

| S-EPMC5150520 | biostudies-literature

CRISPR-Cas9-Guided Genome Engineering in C. elegans.

Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc.

| S-EPMC4932911 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data