Multicolor CRISPR labeling of chromosomal loci in human cells.

Citation data:

Proceedings of the National Academy of Sciences of the United States of America, ISSN: 1091-6490, Vol: 112, Issue: 10, Page: 3002-7

Publication Year:
2015
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Repository URL:
https://escholarship.umassmed.edu/mccb_pubs/10; http://stars.library.ucf.edu/facultybib2010/6680
PMID:
25713381
DOI:
10.1073/pnas.1420024112
PMCID:
PMC4364232
Author(s):
Ma, Hanhui; Naseri, Ardalan; Reyes-Gutierrez, Pablo; Wolfe, Scot A.; Zhang, Shaojie; Pederson, Thoru
Publisher(s):
Proceedings of the National Academy of Sciences
Tags:
Multidisciplinary; Medicine; 4D nucleome; telomeres; pericentromeric DNA; chromosomes; 4D nucleome; telomeres; pericentromeric DNA; chromosomes; DNA-SEQUENCES; CYTOLOGICAL PREPARATIONS; GENE-EXPRESSION; STEM-CELLS; SYSTEM; CAS9; VISUALIZATION; DYNAMICS; TRANSCRIPTION; TALES; Multidisciplinary Sciences; Biochemistry; Cell Biology; Cells; Molecular Biology
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article description
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.