The Nuclear Envelope pp 407-417

Part of the Methods in Molecular Biology book series (MIMB, volume 1411) | Cite as

Visualization of Genomic Loci in Living Cells with a Fluorescent CRISPR/Cas9 System

  • Tobias Anton
  • Heinrich Leonhardt
  • Yolanda Markaki

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.

Key words

CRISPR/Cas9 sgRNA In vivo labeling Repetitive sequences 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Tobias Anton
    • 1
  • Heinrich Leonhardt
    • 1
  • Yolanda Markaki
    • 1
  1. 1.Department of Biology II, BiozentrumLudwig-Maximilians-Universität MünchenPlanegg-MartinsriedGermany

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