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

Author

Listed:
  • Christian H. Gabriel

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

  • Marta Olmo

    (Institute for Theoretical Biology, Charité Universitätsmedizin Berlin)

  • Amin Zehtabian

    (Institute for Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin)

  • Marten Jäger

    (Berlin Institute of Health (BIH) Core Genomics Facility, Charité Universitätsmedizin Berlin)

  • Silke Reischl

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

  • Hannah Dijk

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

  • Carolin Ulbricht

    (Immune Dynamics, Rheumatology and Clinical Immunology, Charité – Universitätsmedizin Berlin
    Immune Dynamics, Deutsches Rheuma-Forschungszentrum (DRFZ), a Leibniz Institute)

  • Asylkhan Rakhymzhan

    (Biophysical Analytics, Deutsches Rheuma-Forschungszentrum (DRFZ), a Leibniz Institute)

  • Thomas Korte

    (Molecular Biophysics, Department of Biology, Humboldt Universität zu Berlin)

  • Barbara Koller

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

  • Astrid Grudziecki

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

  • Bert Maier

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

  • Andreas Herrmann

    (Molecular Biophysics, Department of Biology, Humboldt Universität zu Berlin)

  • Raluca Niesner

    (Biophysical Analytics, Deutsches Rheuma-Forschungszentrum (DRFZ), a Leibniz Institute
    Dynamic and Functional in vivo Imaging, Veterinary Medicine, Freie Universität Berlin)

  • Tomasz Zemojtel

    (Berlin Institute of Health (BIH) Core Genomics Facility, Charité Universitätsmedizin Berlin)

  • Helge Ewers

    (Institute for Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin)

  • Adrián E. Granada

    (Charité Comprehensive Cancer Center, Charité Universitätsmedizin Berlin)

  • Hanspeter Herzel

    (Institute for Theoretical Biology, Charité Universitätsmedizin Berlin)

  • Achim Kramer

    (Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Laboratory of Chronobiology
    Berlin Institute of Health (BIH))

Abstract

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, 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 allowing us 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 (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Suggested Citation

  • Christian H. Gabriel & Marta Olmo & Amin Zehtabian & Marten Jäger & Silke Reischl & Hannah Dijk & Carolin Ulbricht & Asylkhan Rakhymzhan & Thomas Korte & Barbara Koller & Astrid Grudziecki & Bert Maie, 2021. "Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-24086-9
    DOI: 10.1038/s41467-021-24086-9
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    Cited by:

    1. Xu, Yan & Gu, Changgui & Wang, Jiangsheng & Wang, Man & Wang, Haiying & Yang, Huijie & Song, Yuxuan, 2023. "Goodwin oscillator model explains different response of circadian rhythms to constant light," Physica A: Statistical Mechanics and its Applications, Elsevier, vol. 618(C).

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