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

  • Tobias Anton
  • Heinrich Leonhardt
  • Yolanda Markaki
Part of the Methods in Molecular Biology book series (MIMB, volume 1411)


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 



This work was supported by the Deutsche Forschungsgemeinschaft (DFG, SFB 1064 and Nanosystems Initiative Munich, NIM), and T.A. thankfully acknowledges the Graduiertenkolleg GRK1721.


  1. 1.
    Markaki Y, Smeets D, Cremer M, Schermelleh L (2013) Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy. Methods Mol Biol 950:43–64PubMedGoogle Scholar
  2. 2.
    Klug A (2010) The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation. Q Rev Biophys 43:1–21CrossRefPubMedGoogle Scholar
  3. 3.
    Pabo CO, Peisach E, Grant RA (2001) Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem 70:313–340CrossRefPubMedGoogle Scholar
  4. 4.
    Segal DJ, Barbas CF 3rd (2000) Design of novel sequence-specific DNA-binding proteins. Curr Opin Chem Biol 4:34–39CrossRefPubMedGoogle Scholar
  5. 5.
    DeFrancesco L (2011) Move over ZFNs. Nat Biotech 29:681–684CrossRefGoogle Scholar
  6. 6.
    Segal DJ, Dreier B, Beerli RR, Barbas CF 3rd (1999) Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc Natl Acad Sci U S A 96:2758–2763CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Miyanari Y, Ziegler-Birling C, Torres-Padilla ME (2013) Live visualization of chromatin dynamics with fluorescent TALEs. Nat Struct Mol Biol 20:1321–1324CrossRefPubMedGoogle Scholar
  8. 8.
    Ma H, Reyes-Gutierrez P, Pederson T (2013) Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors. Proc Natl Acad Sci U S A 110:21048–21053CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Thanisch K, Schneider K, Morbitzer R, Solovei I, Lahaye T, Bultmann S, Leonhardt H (2014) Targeting and tracing of specific DNA sequences with dTALEs in living cells. Nucleic Acids Res 42, e38CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512CrossRefPubMedGoogle Scholar
  11. 11.
    Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39, e82CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Morbitzer R, Elsaesser J, Hausner J, Lahaye T (2011) Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res 39:5790–5799CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Anton T, Bultmann S, Leonhardt H, Markaki Y (2014) Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system. Nucleus 5:163–172CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ma H, Naseri A, Reyes-Gutierrez P, Wolfe SA, Zhang S, Pederson T (2015) Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A 112:3002–3007CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Westra ER, Swarts DC, Staals RH, Jore MM, Brouns SJ, van der Oost J (2012) The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu Rev Genet 46:311–339CrossRefPubMedGoogle Scholar
  17. 17.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Cremer M, Grasser F, Lanctot C, Muller S, Neusser M, Zinner R, Solovei I, Cremer T (2008) Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol Biol 463:205–239CrossRefPubMedGoogle Scholar
  20. 20.
    Byron M, Hall LL, Lawrence JB (2013) A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures. Curr Protoc Hum Genet Unit 4:15Google Scholar

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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