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 O. Mazzoni and Jane A. Skok ()
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Yi Fu: New York University School of Medicine, 550 First Avenue, Sml 311
Pedro P. Rocha: New York University School of Medicine, 550 First Avenue, Sml 311
Vincent M. Luo: New York University School of Medicine, 550 First Avenue, Sml 311
Ramya Raviram: New York University School of Medicine, 550 First Avenue, Sml 311
Yan Deng: Microscopy Core, New York University School of Medicine
Esteban O. Mazzoni: New York University
Jane A. Skok: New York University School of Medicine, 550 First Avenue, Sml 311

Nature Communications, 2016, vol. 7, issue 1, 1-8

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms11707

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