Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice.

ABSTRACT: CRISPR/dCas9 is a versatile tool that can be used to recruit various effectors and fluorescent molecules to defined genome regions where it can modulate genetic and epigenetic markers, or track the chromatin dynamics in live cells. In vivo applications of CRISPR/dCas9 in animals have been challenged by delivery issues. We generate and characterize a mouse strain with dCas9-EGFP ubiquitously expressed in various tissues. Studying telomere dynamics in these animals reveals surprising results different from those observed in cultured cell lines. The CRISPR/dCas9 knock-in mice provide an important and versatile tool to mechanistically study genome functions in live animals.

SUBMITTER: Duan J

PROVIDER: S-EPMC6225728 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice.

Duan Jinzhi J Lu Guangqing G Hong Yu Y Hu Qingtao Q Mai Xueying X Guo Jing J Si Xiaofang X Wang Fengchao F Zhang Yu Y

Genome biology 20181108 1

CRISPR/dCas9 is a versatile tool that can be used to recruit various effectors and fluorescent molecules to defined genome regions where it can modulate genetic and epigenetic markers, or track the chromatin dynamics in live cells. In vivo applications of CRISPR/dCas9 in animals have been challenged by delivery issues. We generate and characterize a mouse strain with dCas9-EGFP ubiquitously expressed in various tissues. Studying telomere dynamics in these animals reveals surprising results diffe ...[more]

PMID: 30409154

Similar Datasets

Precise modulation of genome functions and live tracking of genome regions in CRISPR/dCas9 knockin mice

Project description:we demonstrated ex vivo and in vivo validation of dCas9 KI functions such as transcription

2017-09-28 | GSE104331 | GEO

Precise modulation of genome functions and live tracking of genome regions in CRISPR/dCas9 knockin mice

Project description:Precise modulation of genome functions and live tracking of genome regions in CRISPR/dCas9 knockin mice

| PRJNA412372 | ENA

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

Project description:The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

| S-EPMC8213786 | biostudies-literature

Genome-wide tracking of dCas9-methyltransferase footprints.

Project description:In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

| S-EPMC5807365 | biostudies-literature

CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.

Project description:Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.

| S-EPMC4894952 | biostudies-literature

Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in.

Project description:In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.

| S-EPMC7452982 | biostudies-literature

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

Project description:Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

| S-EPMC6158506 | biostudies-literature

Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9.

Project description:Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.

| S-EPMC4460908 | biostudies-other

Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

Project description:RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.

| S-EPMC4826288 | biostudies-literature

Tracking of dCas9-methyltransferase footprints

Project description:In normal mammalian development cytosine methylation is essential, and is directed to specific regions of the genome. Despite notable advances in mapping the genome-wide distribution, studying the direct contribution of DNA methylation to gene regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of CRISPR/Cas9 systems with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9-methyltransferase is challenging within a highly modified genome. To overcome this, we utilized an engineered, methylation depleted but maintenance competent mouse ES cell line and find a surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Our results provide new insights into the targeted regulation of DNA methylation and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

2018-03-12 | GSE87757 | GEO

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