title: CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.
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.
authors.affiliation: Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.|Department of Biology, New York University, New York, New York 10003, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.
authors.forename: Pedro P
authors.initials: PP
authors.lastname: Rocha
authors.affiliation: Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.|Department of Biology, New York University, New York, New York 10003, USA.
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Luo
authors.affiliation: Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.|Department of Biology, New York University, New York, New York 10003, USA.
authors.forename: Ramya
authors.initials: R
authors.lastname: Raviram
authors.affiliation: Microscopy Core, New York University School of Medicine, New York, New York 10016, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Deng
authors.affiliation: Department of Biology, New York University, New York, New York 10003, USA.
authors.forename: Esteban O
authors.initials: EO
authors.lastname: Mazzoni
authors.affiliation: Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Skok
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D036022:Capsid Proteins; D004276:DNA, Satellite / Q000032:analysis; D004720:Endonucleases; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D061848:Optical Imaging / Q000379:methods*; D020161:Physical Chromosome Mapping; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D036022:Capsid Proteins; D004276:DNA, Satellite; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases