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Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

Christian H. Gabriel, Marta del Olmo, Amin Zehtabian, Silke Reischl, Hannah van Dijk, Barbara Koller, Astrid Grudziecki, Bert Maier, Helge Ewers, Hanspeter Herzel, Adrian E. Granada, View ORCID ProfileAchim Kramer
doi: https://doi.org/10.1101/2020.02.28.967752
Christian H. Gabriel
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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Marta del Olmo
3Institute for Theoretical Biology, Charité Universitätsmedizin Berlin, Berlin, Germany
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Amin Zehtabian
4Institute for Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany
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Silke Reischl
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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Hannah van Dijk
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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Barbara Koller
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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Astrid Grudziecki
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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Bert Maier
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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Helge Ewers
4Institute for Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany
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Hanspeter Herzel
3Institute for Theoretical Biology, Charité Universitätsmedizin Berlin, Berlin, Germany
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Adrian E. Granada
5IRI Life Sciences, Humboldt-Universität zu Berlin, Berlin, Germany
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Achim Kramer
1Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology, Berlin, Germany
2Berlin Institute of Health (BIH), Berlin, Germany
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  • ORCID record for Achim Kramer
  • For correspondence: achim.kramer@charite.de
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ABSTRACT

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (∼5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator. Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.

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Posted February 28, 2020.
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Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells
Christian H. Gabriel, Marta del Olmo, Amin Zehtabian, Silke Reischl, Hannah van Dijk, Barbara Koller, Astrid Grudziecki, Bert Maier, Helge Ewers, Hanspeter Herzel, Adrian E. Granada, Achim Kramer
bioRxiv 2020.02.28.967752; doi: https://doi.org/10.1101/2020.02.28.967752
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Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells
Christian H. Gabriel, Marta del Olmo, Amin Zehtabian, Silke Reischl, Hannah van Dijk, Barbara Koller, Astrid Grudziecki, Bert Maier, Helge Ewers, Hanspeter Herzel, Adrian E. Granada, Achim Kramer
bioRxiv 2020.02.28.967752; doi: https://doi.org/10.1101/2020.02.28.967752

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