UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; Program in Cell and Developmental Dynamics; Department of Molecular, Cell and Cancer Biology

Date

3-10-2015

Document Type

Article

Disciplines

Biochemistry | Cell Biology | Cells | Molecular Biology

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.

Rights and Permissions

Citation: Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23. Link to article on publisher's site

Comments

Freely available online through the PNAS open access option.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1420024112/-/DCSupplemental.

Related Resources

Link to Article in PubMed

Keywords

4D nucleome, telomeres, pericentromeric DNA, chromosomes

PubMed ID

25713381

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.