title: Visualization of Genomic Loci in Living Cells with a Fluorescent CRISPR/Cas9 System.
abstract: The discovery that the RNA guided bacterial endonuclease Cas9 can be harnessed to target and manipulate user-defined genomic sequences has greatly influenced the field of genome engineering. Interestingly, a catalytically dead Cas9 (dCas9) can be employed as a targeted DNA-binding platform to alter gene expression. By fusing this dCas9 to eGFP, we and others could show that the CRISPR/Cas9 system can be further expanded to label and trace genomic loci in living cells. We demonstrated that by exchanging the sgRNA, dCas9-eGFP could be specifically directed to various heterochromatic sequences within the nucleus. Here, we provide a basic protocol for this versatile tool and describe how to verify new dCas9-eGFP targets.
authors.affiliation: Department of Biology II, Biozentrum, Ludwig-Maximilians-Universität München, Großhadernerstr. 2, 82152, Planegg-Martinsried, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Anton
authors.affiliation: Department of Biology II, Biozentrum, Ludwig-Maximilians-Universität München, Großhadernerstr. 2, 82152, Planegg-Martinsried, Germany. h.leonhardt@lmu.de.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.affiliation: Department of Biology II, Biozentrum, Ludwig-Maximilians-Universität München, Großhadernerstr. 2, 82152, Planegg-Martinsried, Germany.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Markaki
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D002874:Chromosome Mapping / Q000379:methods; D003001:Cloning, Molecular; D015870:Gene Expression; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D016678:Genome; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008856:Microscopy, Fluorescence; D000094704:RNA, Guide, CRISPR-Cas Systems; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas9; In vivo labeling; Repetitive sequences; sgRNA