Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system

Baohui Chen; Luke A Gilbert; Beth A Cimini; Joerg Schnitzbauer; Wei Zhang; Gene-Wei Li; Jason Park; Elizabeth H Blackburn; Jonathan S Weissman; Lei S Qi; Bo Huang

doi:10.1016/j.cell.2013.12.001

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.

Authors

Affiliations

¹ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
² Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, Berkeley, CA 94720, USA.
³ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
⁵ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
⁶ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, Berkeley, CA 94720, USA; California Institute for Quantitative Biomedical Research (QB3), San Francisco, CA 94158, USA.
⁷ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research (QB3), San Francisco, CA 94158, USA. Electronic address: stanley.qi@ucsf.edu.
⁸ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research (QB3), San Francisco, CA 94158, USA. Electronic address: bo.huang@ucsf.edu.

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

Abstract

Publication types

MeSH terms

Substances

Grants and funding