Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1

Mol Cell. 2017 Jan 5;65(1):176-190. doi: 10.1016/j.molcel.2016.11.022. Epub 2016 Dec 22.


To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.

Keywords: Cry1; ES mouse; circadian clock; cryptochrome; knockin mouse; phosphorylation.

MeSH terms

  • Animals
  • Behavior, Animal
  • Circadian Clocks*
  • Circadian Rhythm*
  • Cryptochromes / chemistry
  • Cryptochromes / deficiency
  • Cryptochromes / genetics
  • Cryptochromes / metabolism*
  • Embryonic Stem Cells / metabolism*
  • Genotype
  • HEK293 Cells
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Models, Molecular
  • Motor Activity
  • Mutation
  • NIH 3T3 Cells
  • Period Circadian Proteins / genetics
  • Period Circadian Proteins / metabolism
  • Phenotype
  • Phosphorylation
  • Protein Conformation
  • Signal Transduction
  • Structure-Activity Relationship
  • Time Factors
  • Transfection


  • CRY1 protein, human
  • Cry1 protein, mouse
  • Cry2 protein, mouse
  • Cryptochromes
  • Per2 protein, mouse
  • Period Circadian Proteins