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Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

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 s...

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Autores principales: Dreissig, Steven, Schiml, Simon, Schindele, Patrick, Weiss, Oda, Rutten, Twan, Schubert, Veit, Gladilin, Evgeny, Mette, Michael F., Puchta, Holger, Houben, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599988/
https://www.ncbi.nlm.nih.gov/pubmed/28509419
http://dx.doi.org/10.1111/tpj.13601
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author Dreissig, Steven
Schiml, Simon
Schindele, Patrick
Weiss, Oda
Rutten, Twan
Schubert, Veit
Gladilin, Evgeny
Mette, Michael F.
Puchta, Holger
Houben, Andreas
author_facet Dreissig, Steven
Schiml, Simon
Schindele, Patrick
Weiss, Oda
Rutten, Twan
Schubert, Veit
Gladilin, Evgeny
Mette, Michael F.
Puchta, Holger
Houben, Andreas
author_sort Dreissig, Steven
collection PubMed
description 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.
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spelling pubmed-55999882017-10-02 Live‐cell CRISPR imaging in plants reveals dynamic telomere movements Dreissig, Steven Schiml, Simon Schindele, Patrick Weiss, Oda Rutten, Twan Schubert, Veit Gladilin, Evgeny Mette, Michael F. Puchta, Holger Houben, Andreas Plant J Featured Article (Technical Advance) 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. John Wiley and Sons Inc. 2017-07-14 2017-08 /pmc/articles/PMC5599988/ /pubmed/28509419 http://dx.doi.org/10.1111/tpj.13601 Text en © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Featured Article (Technical Advance)
Dreissig, Steven
Schiml, Simon
Schindele, Patrick
Weiss, Oda
Rutten, Twan
Schubert, Veit
Gladilin, Evgeny
Mette, Michael F.
Puchta, Holger
Houben, Andreas
Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
title Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
title_full Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
title_fullStr Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
title_full_unstemmed Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
title_short Live‐cell CRISPR imaging in plants reveals dynamic telomere movements
title_sort live‐cell crispr imaging in plants reveals dynamic telomere movements
topic Featured Article (Technical Advance)
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599988/
https://www.ncbi.nlm.nih.gov/pubmed/28509419
http://dx.doi.org/10.1111/tpj.13601
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