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Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres
Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fu...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468473/ https://www.ncbi.nlm.nih.gov/pubmed/32973827 http://dx.doi.org/10.3389/fpls.2020.01254 |
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author | Khosravi, Solmaz Schindele, Patrick Gladilin, Evgeny Dunemann, Frank Rutten, Twan Puchta, Holger Houben, Andreas |
author_facet | Khosravi, Solmaz Schindele, Patrick Gladilin, Evgeny Dunemann, Frank Rutten, Twan Puchta, Holger Houben, Andreas |
author_sort | Khosravi, Solmaz |
collection | PubMed |
description | Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. |
format | Online Article Text |
id | pubmed-7468473 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-74684732020-09-23 Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres Khosravi, Solmaz Schindele, Patrick Gladilin, Evgeny Dunemann, Frank Rutten, Twan Puchta, Holger Houben, Andreas Front Plant Sci Plant Science Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. Frontiers Media S.A. 2020-08-20 /pmc/articles/PMC7468473/ /pubmed/32973827 http://dx.doi.org/10.3389/fpls.2020.01254 Text en Copyright © 2020 Khosravi, Schindele, Gladilin, Dunemann, Rutten, Puchta and Houben http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Plant Science Khosravi, Solmaz Schindele, Patrick Gladilin, Evgeny Dunemann, Frank Rutten, Twan Puchta, Holger Houben, Andreas Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres |
title | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres |
title_full | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres |
title_fullStr | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres |
title_full_unstemmed | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres |
title_short | Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres |
title_sort | application of aptamers improves crispr-based live imaging of plant telomeres |
topic | Plant Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468473/ https://www.ncbi.nlm.nih.gov/pubmed/32973827 http://dx.doi.org/10.3389/fpls.2020.01254 |
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