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Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs

  • Quentin R. V. FerryEmail author
  • Tudor A. Fulga
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2162)

Abstract

The type-II CRISPR-Cas9 system has been repurposed to create synthetic programmable transcriptional regulators (CRISPR-TRs). Subsequent modifications of the system now allow for spatiotemporal control of CRISPR-mediated gene activation and repression. Among these solutions, the development of inducible spacer-blocking hairpin guide RNAs (iSBH-sgRNAs) provide an easy to implement and versatile way to condition the activation of most CRISPR-TRs on the presence of a user defined inducer. In this chapter, I cover the know-how relating to the design and synthesis of iSBH-sgRNAs, as well as the implementation in mammalian cells of inducible CRISPR-TR strategies based on this technology.

Key words

CRISPR Cas9 Inducible sgRNA Transcription 

References

  1. 1.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278CrossRefGoogle Scholar
  2. 2.
    Chavez A et al (2016) Comparison of Cas9 activators in multiple species. Nat Methods 13(7):563–567CrossRefGoogle Scholar
  3. 3.
    Ferry QRV, Lyutova R, Fulga TA (2017) Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs. Nat Commun 8:14633CrossRefGoogle Scholar
  4. 4.
    Jain PK et al (2016) Development of light-activated CRISPR using guide RNAs with photocleavable protectors. Angew Chem Int Ed Engl 55(40):12440–12444CrossRefGoogle Scholar
  5. 5.
    Lee YJ et al (2016) Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system. Nucl Acids Res 44(5):gkw056-2473CrossRefGoogle Scholar
  6. 6.
    Liu Y et al (2016) Directing cellular information flow via CRISPR signal conductors. Nat Methods 66(4):1173–1179Google Scholar
  7. 7.
    Tang W, Hu JH, Liu DR (2017) Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation. Nat Commun 8:15939CrossRefGoogle Scholar
  8. 8.
    Baeumler TA, Ahmed AA, Fulga TA (2017) Engineering synthetic signaling pathways with programmable dCas9-based chimeric receptors. Cell Rep 20(11):2639–2653CrossRefGoogle Scholar
  9. 9.
    Aricescu AR, Lu W, Jones EY (2006) A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62(Pt 10):1243–1250CrossRefGoogle Scholar
  10. 10.
    Untergasser A et al (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15):e115–e115CrossRefGoogle Scholar
  11. 11.
    Bennett CF, Swayze EE (2010) RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol 50(1):259–293CrossRefGoogle Scholar
  12. 12.
    Zheng C, Baum BJ (2008) Evaluation of promoters for use in tissue-specific gene delivery. Methods Mol Biol (Clifton, NJ) 434(Chapter 13):205–219Google Scholar
  13. 13.
    Gossen M et al (1995) Transcriptional activation by tetracyclines in mammalian cells. Science (New York, NY) 268(5218):1766–1769CrossRefGoogle Scholar
  14. 14.
    Gilbert LA et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451CrossRefGoogle Scholar
  15. 15.
    Mali P et al (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31(9):833–838CrossRefGoogle Scholar
  16. 16.
    Konermann S et al (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588CrossRefGoogle Scholar
  17. 17.
    Chavez A et al (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12(4):326–328CrossRefGoogle Scholar
  18. 18.
    Gilbert LA et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159(3):647–661CrossRefGoogle Scholar
  19. 19.
    Shechner DM et al (2015) Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat Methods 12(7):664–670CrossRefGoogle Scholar
  20. 20.
    Zalatan JG et al (2014) Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160(1-2):339–350CrossRefGoogle Scholar
  21. 21.
    Zadeh JN et al (2011) NUPACK: analysis and design of nucleic acid systems. J Comput Chem 32(1):170–173CrossRefGoogle Scholar
  22. 22.
    Dahlman JE et al (2015) Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat Biotechnol 33(11):1159–1161CrossRefGoogle Scholar
  23. 23.
    Kiani S et al (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 12(11):1051–1054CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Radcliffe Department of Medicine, Weatherall Institute of Molecular MedicineUniversity of OxfordOxfordUK

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