Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001.

Abstract

The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Genetic Techniques*
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • In Situ Hybridization, Fluorescence
  • Karyotyping
  • Mitosis
  • Molecular Sequence Data
  • Mucin-4 / genetics
  • Telomere*

Substances

  • MUC4 protein, human
  • Mucin-4
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins