Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells
| Autor: | Gabriel, Christian H., del Olmo, Marta, Zehtabian, Amin, Jäger, Marten, Reischl, Silke, van Dijk, Hannah, Ulbricht, Carolin, Rakhymzhan, Asylkhan, Korte, Thomas, Koller, Barbara, Grudziecki, Astrid, Maier, Bert, Herrmann, Andreas, Niesner, Raluca, Zemojtel, Tomasz, Ewers, Helge, Granada, Adrián E., Herzel, Hanspeter, Kramer, Achim |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Cell biology
endocrine system CRY1 animal structures Science CLOCK Proteins Article Genes Reporter Cell Line Tumor Circadian Clocks Humans Gene Knock-In Techniques fungi Biological techniques CRISPR technology PER2 Period Circadian Proteins HCT116 Cells Circadian Rhythm Cryptochromes sense organs Cell biology of circadian clocks CRISPR-Cas Systems Single-Cell Analysis 600 Technik Medizin angewandte Wissenschaften::610 Medizin und Gesundheit::610 Medizin und Gesundheit |
| Zdroj: | Nature Communications, Vol 12, Iss 1, Pp 1-15 (2021) Nature Communications |
| DOI: | 10.1038/s41467-021-24086-9 |
| Popis: | 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. Live-cell recordings have been an important tool for studying circadian rhythms. Here the authors use CRISPR gene editing mediated knock-in to fluorescently tag Per2 and Cry1, and study cellular circadian dynamics of these two clock proteins. |
| Databáze: | OpenAIRE |
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