Summary Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.
Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
Steven Dreissig,Simon Schiml,Patrick Schindele,Oda Weiss,T. Rutten,V. Schubert,E. Gladilin,M. Mette,H. Puchta,A. Houben
Published 2017 in The Plant Journal
ABSTRACT
PUBLICATION RECORD
- Publication year
2017
- Venue
The Plant Journal
- Publication date
2017-07-14
- Fields of study
Biology, Medicine, Environmental Science
- Identifiers
- External record
- Source metadata
Semantic Scholar, PubMed
CITATION MAP
EXTRACTION MAP
CLAIMS
CONCEPTS
- crispr-dcas9 imaging
A live-cell imaging approach that uses catalytically inactive Cas9 proteins guided by CRISPR RNAs to label genomic DNA.
Aliases: CRISPR imaging, dCas9 imaging
- dna-protein interactions in vivo
Physical associations between DNA and proteins inside living plant cells that can be visualized by co-imaging.
Aliases: DNA-protein interaction
- dual dcas9 orthologues
The use of two catalytically inactive Cas9 proteins from different bacterial species in the same imaging setup.
Aliases: two dCas9 orthologues
- fluorescence-labelled proteins
Proteins tagged with fluorescent markers so they can be imaged alongside the DNA-targeting CRISPR constructs.
Aliases: fluorescently labelled proteins, fluorescent-labeled proteins
- fluorescent reporters
Fluorescent protein tags, including eGFP and mRuby2, used to make the dCas9 constructs visible in live cells.
Aliases: eGFP, mRuby2, fluorescent labels
- interphase
The non-dividing stage of the cell cycle during which the telomere dynamics were tracked.
- live leaf cells of nicotiana benthamiana
Living leaf cells from the model plant Nicotiana benthamiana used for the in vivo imaging experiments.
Aliases: Nicotiana benthamiana leaf cells, leaf cells
- live plant cells
Living cells from plants used as the biological context for the CRISPR imaging experiments.
Aliases: plant cells
- multiple genomic loci
More than one distinct genomic position that can be targeted or visualized within the same cell.
Aliases: multiple loci
- staphylococcus aureus cas9
The Cas9 orthologue from Staphylococcus aureus used here as a catalytically inactive DNA-binding scaffold.
Aliases: SaCas9
- streptococcus pyogenes cas9
The Cas9 orthologue from Streptococcus pyogenes used here as a catalytically inactive DNA-binding scaffold.
Aliases: SpCas9
- telomere movement
Changes in the nuclear position of telomere signals over time as measured by live-cell imaging.
Aliases: telomere dynamics
- telomere repeats
Repetitive DNA sequences at chromosome ends that serve as the genomic targets in the imaging experiments.
Aliases: telomeres
REFERENCES
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