title: Multicolor CRISPR labeling of chromosomal loci in human cells.
abstract: The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.
authors.affiliation: Program in Cell and Developmental Dynamics and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; hanhui.ma@umassmed.edu thoru.pederson@umassmed.edu.
authors.forename: Hanhui
authors.initials: H
authors.lastname: Ma
authors.affiliation: Department of Electrical Engineering and Computer Science, University of Central Florida, Orlando, FL 32816; and.
authors.forename: Ardalan
authors.initials: A
authors.lastname: Naseri
authors.affiliation: Program in Cell and Developmental Dynamics and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605;
authors.forename: Pablo
authors.initials: P
authors.lastname: Reyes-Gutierrez
authors.affiliation: Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605.
authors.forename: Scot A
authors.initials: SA
authors.lastname: Wolfe
authors.affiliation: Department of Electrical Engineering and Computer Science, University of Central Florida, Orlando, FL 32816; and.
authors.forename: Shaojie
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Program in Cell and Developmental Dynamics and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; hanhui.ma@umassmed.edu thoru.pederson@umassmed.edu.
authors.forename: Thoru
authors.initials: T
authors.lastname: Pederson
mesh_terms: D045744:Cell Line, Tumor; D002874:Chromosome Mapping; D002877:Chromosomes, Human; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D006801:Humans; D008856:Microscopy, Fluorescence
keywords: 4D nucleome; chromosomes; pericentromeric DNA; telomeres