title: Imaging Specific Genomic DNA in Living Cells.
abstract: The three-dimensional organization of the genome plays important roles in regulating the functional output of the genome and even in the maintenance of epigenetic inheritance and genome stability. Here, we review and compare a number of newly developed methods-especially those that utilize the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system-that enable the direct visualization of specific, endogenous DNA sequences in living cells. We also discuss the practical considerations in implementing the CRISPR imaging technique to achieve sufficient signal-to-background levels, high specificity, and high labeling efficiency. These DNA labeling methods enable tracking of the copy number, localization, and movement of genomic elements, and we discuss the potential applications of these methods in understanding the searching and targeting mechanism of the Cas9-sgRNA complex, investigating chromosome organization, and visualizing genome instability and rearrangement.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; email: baohui.chen@ucsf.edu , juan.guan@ucsf.edu , bo.huang@ucsf.edu.
authors.forename: Baohui
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; email: baohui.chen@ucsf.edu , juan.guan@ucsf.edu , bo.huang@ucsf.edu.
authors.forename: Juan
authors.initials: J
authors.lastname: Guan
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; email: baohui.chen@ucsf.edu , juan.guan@ucsf.edu , bo.huang@ucsf.edu.
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D016678:Genome; D006801:Humans; D057054:Molecular Imaging / Q000379:methods
chemical_list: D064130:CRISPR-Associated Proteins; D004247:DNA
keywords: CRISPR; Cas9; chromatin; fluorescence; microscopy; nucleus