Effect of Multiple Clock Gene Ablations on the Circadian Period Length and Temperature Compensation in Mammalian Cells.
Most organisms have cell-autonomous circadian clocks to coordinate their activity and physiology according to 24-h environmental changes. Despite recent progress in circadian studies, it is not fully understood how the period length and the robustness of mammalian circadian rhythms are determined. In this study, we established a series of mouse embryonic stem cell (ESC) lines with single or multiplex clock gene ablations using the CRISPR/Cas9-based genome editing method. ESC-based in vitro circadian clock formation assay shows that the CRISPR-mediated clock gene disruption not only reproduces the intrinsic circadian molecular rhythms of previously reported mice tissues and cells lacking clock genes but also reveals that complexed mutations, such as CKIδ(m/m):CKIε(+/m):Cry2(m/m) mutants, exhibit an additively lengthened circadian period. By using these mutant cells, we also investigated the relation between period length alteration and temperature compensation. Although CKIδ-deficient cells slightly affected the temperature insensitivity of period length, we demonstrated that the temperature compensation property is largely maintained in all mutants. These results show that the ESC-based assay system could offer a more systematic and comprehensive approach to the genotype-chronotype analysis of the intracellular circadian clockwork in mammals.
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Yoshiki
Y
Tsuchiya
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Yasuhiro
Y
Umemura
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Yoichi
Y
Minami
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Nobuya
N
Koike
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan Department of Orthopedic Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Toshihiro
T
Hosokawa
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan Department of Nephrology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Masayuki
M
Hara
Faculty of Design, Kyushu University, Fukuoka, Japan.
Hiroshi
H
Ito
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Hitoshi
H
Inokawa
Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan kyagita@koto.kpu-m.ac.jp.
Kazuhiro
K
Yagita
D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D057906:Circadian Clocks / Q000235:genetics*; D002940:Circadian Rhythm / Q000235:genetics*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism; D009154:Mutation; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D056950:Period Circadian Proteins
CRISPR/Cas9; clock genes; embryonic stem cells; period length; temperature compensation