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...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-11-01
|
| Series: | Biology |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-7737/10/11/1204 |
| _version_ | 1827677346411315200 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T05:41:47Z |
| format | Article |
| id | doaj.art-f2ed6292f0784bc795ee4c1e1092cfbf |
| institution | Directory Open Access Journal |
| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-10T05:41:47Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biology |
| spelling | doaj.art-f2ed6292f0784bc795ee4c1e1092cfbf2023-11-22T22:28:53ZengMDPI AGBiology2079-77372021-11-011011120410.3390/biology10111204Systematic Studies of the Circadian Clock Genes Impact on Temperature Compensation and Cell Proliferation Using CRISPR ToolsYue Wu0Tian Tian1Yin Wu2Yu Yang3Yunfei Zhang4Ximing Qin5Department of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, ChinaDepartment of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, ChinaDepartment of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, ChinaDepartment of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, ChinaDepartment of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, ChinaDepartment of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, ChinaMammalian 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.https://www.mdpi.com/2079-7737/10/11/1204circadian rhythmclock genestemperature compensationgenetic editingCRISPR |
| spellingShingle | 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 Biology circadian rhythm clock genes temperature compensation genetic editing CRISPR |
| title | 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_short | 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 |
| topic | circadian rhythm clock genes temperature compensation genetic editing CRISPR |
| url | https://www.mdpi.com/2079-7737/10/11/1204 |
| work_keys_str_mv | AT yuewu systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT tiantian systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT yinwu systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT yuyang systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT yunfeizhang systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools AT ximingqin systematicstudiesofthecircadianclockgenesimpactontemperaturecompensationandcellproliferationusingcrisprtools |