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Light-Inducible CRISPR Labeling

Part of the Methods in Molecular Biology book series (MIMB,volume 2173)

Abstract

CRISPR labeling is a powerful technique to study the chromatin architecture in live cells. In CRISPR labeling, a catalytically dead CRISPR-Cas9 mutant is employed as programmable DNA-binding domain to recruit fluorescent proteins to selected genomic loci. The fluorescently labeled loci can then be identified as fluorescent spots and tracked over time by microscopy. A limitation of this approach is the lack of temporal control of the labeling process itself: Cas9 binds to the g(uide)RNA-complementary target loci as soon as it is expressed. The decoration of the genome with Cas9 molecules will, however, interfere with gene regulation and—possibly—affect the genome architecture itself. The ability to switch on and off Cas9 DNA binding in CRISPR labeling experiments would thus be important to enable more precise interrogations of the chromatin spatial organization and dynamics and could further be used to study Cas9 DNA binding kinetics directly in living human cells.

Here, we describe a detailed protocol for light-inducible CRISPR labeling. Our method employs CASANOVA, an engineered, optogenetic anti-CRISPR protein, which efficiently traps the Streptococcus pyogenes (Spy)Cas9 in the dark, but permits Cas9 DNA targeting upon illumination with blue light. Using telomeres as exemplary target loci, we detail the experimental steps required for inducible CRISPR labeling with CASANOVA. We also provide instructions on how to analyze the resulting microscopy data in a fully automated fashion.

Key words

  • CRISPR labeling
  • Telomere
  • LOV2 domain
  • Photoreceptor
  • Anti-CRISPR
  • CASANOVA
  • Automated image analysis
  • KNIME

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References

  1. Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/10.1126/science.1225829

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  2. Mali et al (2013) Science, 2013: RNA-guided human genome engineering via Cas9. Full citation can be found here: https://www.ncbi.nlm.nih.gov/pubmed/23287722

  3. Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Mali P, Aach J, Stranges PB et al (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31 (9):833–838. https://doi.org/10.1038/nbt.2675

  5. Gilbert LA, Larson MH, Morsut L et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451. https://doi.org/10.1016/j.cell.2013.06.044

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Cheng AW, Wang H, Yang H et al (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23(10):1163–1171. https://doi.org/10.1038/cr.2013.122

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Perez-Pinera P, Kocak DD, Vockley CM et al (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10(10):973–976. https://doi.org/10.1038/nmeth.2600

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Hilton IB, D’Ippolito AM, Vockley CM et al (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33(5):510–517. https://doi.org/10.1038/nbt.3199

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Liu XS, Wu H, Ji X et al (2016) Editing DNA methylation in the mammalian genome. Cell 167(1):233–247.e217. https://doi.org/10.1016/j.cell.2016.08.056

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Komor AC, Kim YB, Packer MS et al (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603):420–424. https://doi.org/10.1038/nature17946

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Gaudelli NM, Komor AC, Rees HA et al (2017) Programmable base editing of A∗T to G∗C in genomic DNA without DNA cleavage. Nature 551(7681):464–471. https://doi.org/10.1038/nature24644

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Chen BH, Gilbert LA, Cimini BA et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155(7):1479–1491. https://doi.org/10.1016/j.cell.2013.12.001

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  13. Tanenbaum ME, Gilbert LA, Qi LS et al (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159(3):635–646. https://doi.org/10.1016/j.cell.2014.09.039

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  14. Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. https://doi.org/10.1016/j.cell.2013.02.022

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  15. Bubeck F, Hoffmann MD, Harteveld Z et al (2018) Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat Methods 15(11):924–927. https://doi.org/10.1038/s41592-018-0178-9

    CAS  CrossRef  PubMed  Google Scholar 

  16. Rauch BJ, Silvis MR, Hultquist JF et al (2017) Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168(1–2):150–158.e110. https://doi.org/10.1016/j.cell.2016.12.009

    CAS  CrossRef  PubMed  Google Scholar 

  17. Ma H, Naseri A, Reyes-Gutierrez P et al (2015) Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A 112(10):3002–3007. https://doi.org/10.1073/pnas.1420024112

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  18. Ma H, Tu LC, Naseri A et al (2016) Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol 34(5):528–530. https://doi.org/10.1038/nbt.3526

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  19. Pawluk A, Amrani N, Zhang Y et al (2016) Naturally occurring Off-Switches for CRISPR-Cas9. Cell 167(7):1829–1838.e1829. https://doi.org/10.1016/j.cell.2016.11.017

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

M.D.H. was supported by a Helmholtz International Graduate School for Cancer Research scholarship (DKFZ, Heidelberg).

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Correspondence to Dominik Niopek .

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Hoffmann, M.D., Bubeck, F., Niopek, D. (2020). Light-Inducible CRISPR Labeling. In: Niopek, D. (eds) Photoswitching Proteins . Methods in Molecular Biology, vol 2173. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0755-8_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0755-8_9

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  • Publisher Name: Humana, New York, NY

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