CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci

Yi Fu, Pedro P. Rocha, Vincent M. Luo, Ramya Raviram, Yan Deng, Esteban Mazzoni, Jane A. Skok

Research output: Contribution to journalArticle

Abstract

Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.

Original languageEnglish (US)
Article number11707
JournalNature Communications
Volume7
DOIs
StatePublished - Jan 1 2016

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Viral RNA
loci
Scaffolds
Imaging systems
Labeling
Visualization
Guide RNA
Color
Genetic Epigenesis
Satellites
color
Imaging techniques
Organizational Innovation
Chromosomes, Human, Pair 12
marking
RNA-Binding Proteins
Viral Proteins
Chromosomes
Gene expression

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

Cite this

CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. / Fu, Yi; Rocha, Pedro P.; Luo, Vincent M.; Raviram, Ramya; Deng, Yan; Mazzoni, Esteban; Skok, Jane A.

In: Nature Communications, Vol. 7, 11707, 01.01.2016.

Research output: Contribution to journalArticle

Fu, Yi ; Rocha, Pedro P. ; Luo, Vincent M. ; Raviram, Ramya ; Deng, Yan ; Mazzoni, Esteban ; Skok, Jane A. / CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. In: Nature Communications. 2016 ; Vol. 7.
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