title: Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.
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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Baohui
authors.initials: B
authors.lastname: Chen
authors.affiliation: 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.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Gilbert
authors.affiliation: 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.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Cimini
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Joerg
authors.initials: J
authors.lastname: Schnitzbauer
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Gene-Wei
authors.initials: GW
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Jason
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Elizabeth H
authors.initials: EH
authors.lastname: Blackburn
authors.affiliation: 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.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
authors.affiliation: 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.
authors.forename: Lei S
authors.initials: LS
authors.lastname: Qi
authors.affiliation: 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.
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
mesh_terms: D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D008938:Mitosis; D008969:Molecular Sequence Data; D055264:Mucin-4 / Q000235:genetics; D016615:Telomere
chemical_list: C524658:MUC4 protein, human; D055264:Mucin-4; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins