Entwicklung einer CRISPR-FISH Methode zur Sichtbarmachung genomischer Sequenzen in lebenden Pflanzen und ihrer Anwendung bei der Analyse der Zentromerstruktur und dem Prozess der Chromosomen Kondensation in lebenden Zellen
Zusammenfassung der Projektergebnisse
To establish a CRISPR/dCas9-based live cell imaging for plants, two catalytically inactive Cas9 orthologues of Streptococcus pyogenes (Sp-dCas9) and Staphylococcus aureus (SadCas9) were applied. Multiple copies of fluorescence proteins, either eGFP or mRuby2, were fused to the C-terminal end of each dCas9 variant. A robust visualization of telomere repeats of transiently transformed Nicotiana benthamiana revealed dynamic telomere movements of up to 2 µm within 30 minutes during interphase. Furthermore, CRISPR/dCas9-imaging can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we paved the way for imaging of multiple genomic loci in live plants cells. In order to improve the intensity of the reporter construct the sgRNA scaffold was fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the aptamer-binding proteins fused to fluorescent protein (MCP-FP and PCP-FP) are recruited to the targeted sequence. Compared to dCas9:GFP, the quality of telomere labelling was improved in transiently transformed N. benthamiana. Labelling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labelling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. A novel tool was developed to visualize defined genomic sequences in fixed plant and animal nuclei and chromosomes based on a two-part guide RNA recombinant Cas9 endonuclease complex. In contrast to classical FISH, RGEN-ISL (RNA-guided endonuclease – in situ labelling) does not require DNA denaturation and permits a better structural chromatin preservation. The application of differentially labelled tracrRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry, FISH and DNA replication studies. Real-time visualization 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 revolutionize the field of chromosome biology.
Projektbezogene Publikationen (Auswahl)
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2017. Live-cell CRISPR imaging in plants reveals dynamic telomere movements. Plant J 91, 565-573
Dreissig S, Schiml S, Schindele P, Weiss O, Rutten T, Schubert V, Gladilin E, Mette MF, Puchta H, Houben A
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Live imaging of chromatin by CRISPR in plant cells. 02.09-05.09. 2018, 22nd International Chromosome Conference (ICC), Prague, Czech Republic
Solmaz Khosravi, Steven Dreissig, Patrick Schindele, Holger Puchta, Andreas Houben
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(2019) CRISPR/Cas9- based RGEN-ISL allows the simultaneous and specific visualization of proteins, DNA repeats, and sites of DNA replication. Cytogenet Genome Res 159 (1):48-53
Nemeckova A, Wasch C, Schubert V, Ishii T, Hribova E, Houben A
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(2019) RNA-guided endonuclease - in situ labelling (RGEN-ISL): a fast CRISPR/Cas9-based method to label genomic sequences in various species. New Phytol 222 (3):1652-1661
Ishii T, Schubert V, Khosravi S, Dreissig S, Metje-Sprink J, Sprink T, Fuchs J, Meister A, Houben A
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(2020) Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells. Chromosome Res 28 (1):7-17
Khosravi S, Ishii T, Dreissig S, Houben A
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(2020) Application of aptamers improves CRISPR-based live imaging of plant telomeres. Frontiers in Plant Sciences
Khosravi, Schindele, Gladilin, Dunemann, Rutten, Puchta, Houben
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(2020) Application of Tris-HCl allows the specific labeling of regularly prepared chromosomes by CRISPR-FISH. Cytogenet Genome Res. 160: 156-165
Potlapalli BP, Schubert V, Metje-Sprink J, Liehr T, Houben A
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(2020) Live Cell CRISPR imaging in plant cells with a telomere-specific guide RNA. In Book: RNA Tagging Methods and Protocols Editors: Heinlein, Manfred (Ed.) Springer, ISBN 978-1-0716- 0711-4
Khosravi, Dreissig, Schindele, Wolter, Rutten, Puchta, Houben
