Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

Nat Commun. 2021 Jun 18;12(1):3796. doi: 10.1038/s41467-021-24086-9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CLOCK Proteins / metabolism*
  • CRISPR-Cas Systems / genetics
  • Cell Line, Tumor
  • Circadian Clocks / physiology*
  • Circadian Rhythm / physiology
  • Cryptochromes / genetics
  • Cryptochromes / metabolism*
  • Gene Knock-In Techniques / methods
  • Genes, Reporter / genetics
  • HCT116 Cells
  • Humans
  • Period Circadian Proteins / genetics
  • Period Circadian Proteins / metabolism*
  • Single-Cell Analysis / methods*

Substances

  • CRY1 protein, human
  • Cryptochromes
  • PER2 protein, human
  • Period Circadian Proteins
  • CLOCK Proteins
  • CLOCK protein, human