CRISPR-dCas9 and SgRNA Scaffolds Enable Dual-colour Live Imaging of Satellite Sequences and Repeat-enriched Individual Loci

Overview

Journal Nat Commun

Specialty Biology

Date 2016 May 26

PMID 27222091

Citations 75

Authors

Yi Fu

Pedro P Rocha

Vincent M Luo

Ramya Raviram

Yan Deng

Esteban O Mazzoni

Jane A Skok

Affiliations

Soon will be listed here.

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.

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