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Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.

Multicolor CRISPR labeling of chromosomal loci in human cells.

Author information

1
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.
2
Department of Electrical Engineering and Computer Science, University of Central Florida, Orlando, FL 32816; and.
3
Program in Cell and Developmental Dynamics and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605;
4
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605.

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.

KEYWORDS:

4D nucleome; chromosomes; pericentromeric DNA; telomeres

PMID:
25713381
PMCID:
PMC4364232
DOI:
10.1073/pnas.1420024112
[Indexed for MEDLINE]
Free PMC Article

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