SUMMARY In contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics in interphase are less understood. We developed a sensitive, multicolor system, CRISPR-Sirius, allowing the real-time tracking of the dynamics of chromosomal loci. We tracked loci kilobases to megabases apart and found significant variation in the inter-locus distances of each pair, indicating differing degrees of DNA contortion. We resolved two distinct modes of dynamics of loci: saltatory local movements as well as translational movements of the domain. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle-dependence are indicative of a hitherto unrecognized compaction-relaxation dynamic of the chromosomal fiber operating concurrently with changes in the extent of observed genomic domain movements. IN BRIEF Distinct chromosome folding and dynamics during cell cycle progression were dissected by CRISPR-Sirius DNA imaging in living cells. HIGHLIGHTS CRISPR-Sirius allows tracking of pairs of chromosomal loci having kilobase to megabase inter-locus distances Pair-wise tracking of loci allows measurement of both local and domain dynamics Chromosomal fiber relaxation is positively correlated with local dynamics Genomic region size contributes to local and domain movements Distinct chromosome dynamics were uncovered during cell cycle progression in interphase

CRISPR-Based DNA Imaging in Living Cells Reveals Cell Cycle-Dependent Chromosome Dynamics