Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools

Mammalian circadian genes are capable of producing a self-sustained, autonomous oscillation whose period is around 24 h. One of the major characteristics of the circadian clock is temperature compensation. However, the mechanism underlying temperature compensation remains elusive. Previous studies i...

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Autores principales:	Yue Wu, Tian Tian, Yin Wu, Yu Yang, Yunfei Zhang, Ximing Qin
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	circadian rhythm clock genes temperature compensation genetic editing CRISPR Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/f2ed6292f0784bc795ee4c1e1092cfbf
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spelling	oai:doaj.org-article:f2ed6292f0784bc795ee4c1e1092cfbf2021-11-25T16:48:03ZSystematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools10.3390/biology101112042079-7737https://doaj.org/article/f2ed6292f0784bc795ee4c1e1092cfbf2021-11-01T00:00:00Zhttps://www.mdpi.com/2079-7737/10/11/1204https://doaj.org/toc/2079-7737Mammalian circadian genes are capable of producing a self-sustained, autonomous oscillation whose period is around 24 h. One of the major characteristics of the circadian clock is temperature compensation. However, the mechanism underlying temperature compensation remains elusive. Previous studies indicate that a single clock gene may determine the temperature compensation in several model organisms. In order to understand the influence of each individual clock gene on the temperature compensation, twenty-three well-known mammalian clock genes plus <i>Timeless</i> and <i>Myc</i> genes were knocked out individually, using a powerful gene-editing tool, CRISPR/Cas9. First, <i>Bmal1</i>, <i>Cry1</i>, and <i>Cry2</i> were knocked out as examples to verify that deleting genes by CRISPR is effective and precise. Cell lines targeting twenty-two genes were successfully edited in mouse fibroblast NIH3T3 cells, and off-target analysis indicated these genes were correctly knocked out. Through measuring the luciferase reporters, the circadian periods of each cell line were recorded under two different temperatures, 32.5 °C and 37 °C. The temperature compensation coefficient Q<sub>10</sub> was subsequently calculated for each cell line. Estimations of the Q<sub>10</sub> of these cell lines showed that none of the individual cell lines can adversely affect the temperature compensation. Cells with a longer period at lower temperature tend to have a shorter period at higher temperature, while cells with a shorter period at lower temperature tend to be longer at higher temperature. Thus, the temperature compensation is a fundamental property to keep cellular homeostasis. We further conclude that the temperature compensation is a complex gene regulation system instead of being regulated by any single gene. We also estimated the proliferation rates of these cell lines. After systematically comparing the proliferation rates and circadian periods, we found that the cell growth rate is not dependent on the circadian period.Yue WuTian TianYin WuYu YangYunfei ZhangXiming QinMDPI AGarticlecircadian rhythmclock genestemperature compensationgenetic editingCRISPRBiology (General)QH301-705.5ENBiology, Vol 10, Iss 1204, p 1204 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	circadian rhythm clock genes temperature compensation genetic editing CRISPR Biology (General) QH301-705.5
spellingShingle	circadian rhythm clock genes temperature compensation genetic editing CRISPR Biology (General) QH301-705.5 Yue Wu Tian Tian Yin Wu Yu Yang Yunfei Zhang Ximing Qin Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools
description	Mammalian circadian genes are capable of producing a self-sustained, autonomous oscillation whose period is around 24 h. One of the major characteristics of the circadian clock is temperature compensation. However, the mechanism underlying temperature compensation remains elusive. Previous studies indicate that a single clock gene may determine the temperature compensation in several model organisms. In order to understand the influence of each individual clock gene on the temperature compensation, twenty-three well-known mammalian clock genes plus <i>Timeless</i> and <i>Myc</i> genes were knocked out individually, using a powerful gene-editing tool, CRISPR/Cas9. First, <i>Bmal1</i>, <i>Cry1</i>, and <i>Cry2</i> were knocked out as examples to verify that deleting genes by CRISPR is effective and precise. Cell lines targeting twenty-two genes were successfully edited in mouse fibroblast NIH3T3 cells, and off-target analysis indicated these genes were correctly knocked out. Through measuring the luciferase reporters, the circadian periods of each cell line were recorded under two different temperatures, 32.5 °C and 37 °C. The temperature compensation coefficient Q<sub>10</sub> was subsequently calculated for each cell line. Estimations of the Q<sub>10</sub> of these cell lines showed that none of the individual cell lines can adversely affect the temperature compensation. Cells with a longer period at lower temperature tend to have a shorter period at higher temperature, while cells with a shorter period at lower temperature tend to be longer at higher temperature. Thus, the temperature compensation is a fundamental property to keep cellular homeostasis. We further conclude that the temperature compensation is a complex gene regulation system instead of being regulated by any single gene. We also estimated the proliferation rates of these cell lines. After systematically comparing the proliferation rates and circadian periods, we found that the cell growth rate is not dependent on the circadian period.
format	article
author	Yue Wu Tian Tian Yin Wu Yu Yang Yunfei Zhang Ximing Qin
author_facet	Yue Wu Tian Tian Yin Wu Yu Yang Yunfei Zhang Ximing Qin
author_sort	Yue Wu
title	Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools
title_short	Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools
title_full	Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools
title_fullStr	Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools
title_full_unstemmed	Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools
title_sort	systematic studies of the circadian clock genes impact on temperature compensation and cell proliferation using crispr tools
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/f2ed6292f0784bc795ee4c1e1092cfbf
work_keys_str_mv	AT yuewu systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT tiantian systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT yinwu systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT yuyang systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT yunfeizhang systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT ximingqin systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools
_version_	1718412993233944576

Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR Tools

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