RNA-guided endonuclease - in situ labelling (RGEN-ISL) : a fast CRISPR/Cas9-based method to label genomic sequences in various species
© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.
| Veröffentlicht in: | The New phytologist. - 1979. - 222(2019), 3 vom: 26. Mai, Seite 1652-1661 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2019
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| Zugriff auf das übergeordnete Werk: | The New phytologist |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't FISH CRISPR/Cas9 centromere immunostaining in situ labelling telomere RNA, Guide, CRISPR-Cas Systems CRISPR-Associated Protein 9 mehr... |
| Zusammenfassung: | © 2019 The Authors. New Phytologist © 2019 New Phytologist Trust. Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology |
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| Beschreibung: | Date Completed 27.02.2020 Date Revised 04.01.2024 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1469-8137 |
| DOI: | 10.1111/nph.15720 |