CpG Islands pp 285-301 | Cite as

Functional Insulator Scanning of CpG Islands to Identify Regulatory Regions of Promoters Using CRISPR

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1766)

Abstract

The ability to mutate a promoter in situ is potentially a very useful approach for gaining insights into endogenous gene regulation mechanisms. The advent of CRISPR/Cas systems has provided simple, efficient, and targeted genetic manipulation in eukaryotes, which can be applied to studying genome structure and function.

The basic CRISPR toolkit comprises an endonuclease, Cas9, and a short DNA-targeting sequence, made up of a single guide RNA (sgRNA). The catalytic domains of Cas9 are rendered active upon dimerization of Cas9 with sgRNA, resulting in targeted double stranded DNA breaks. Among other applications, this method of DNA cleavage can be coupled to endogenous homology-directed repair (HDR) mechanisms for the generation of site-specific editing or knockin mutations, at both promoter regulatory and gene coding sequences.

A well-characterized regulatory feature of promoter regions is the high abundance of CpGs. These CpG islands tend to be unmethylated, ensuring a euchromatic environment that promotes gene transcription. Here, we demonstrate CRISPR-mediated editing of two CpG islands located within the promoter region of the MDR1 gene (Multi Drug Resistance 1). Cas9 is used to generate double stranded breaks across multiple target sites, which are then repaired while inserting the beta globin (β-globin) insulator, 5′HS5. Thus, we are screening through promoter regulatory sequences with a chromatin barrier element to identify functional regions via “insulator scanning.” Transcriptional and functional assessment of MDR1 expression provides evidence of genome engineering. Overall, this method allows the scanning of CpG islands to identify their promoter functions.

Key words

Genome engineering CRISPR CpG islands DNA methylation Insulator scanning MDR1 

Notes

Acknowledgments

The authors were funded by Wellcome Trust UK New Investigator Award No. WT102944 (MI, AG) and a BBSRC-Innovate UK Industrial Biotechnology Catalyst Grant BB/M028933/1 (MM). Alice Grob and Masue Marbiah contributed equally to this work.

References

  1. 1.
    Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wang T, Wei JJ, Sabatini DM et al (2013) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166):80–84CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Shan Q, Wang Y, Li J et al (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31(8):686–688CrossRefPubMedGoogle Scholar
  4. 4.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821CrossRefPubMedGoogle Scholar
  5. 5.
    Gasiunas G, Barrangou R, Horvath P et al (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109(39):E2579–E2586CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Sternberg SH, Redding S, Jinek M et al (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507(7490):62–67CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Chen B, 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):1479–1491CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Shrivastav M, De Haro LP, Nickoloff JA (2008) Regulation of DNA double-strand break repair pathway choice. Cell Res 18(1):134–147CrossRefPubMedGoogle Scholar
  9. 9.
    McDonald JI, Celik H, Rois LE et al (2016) Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol Open 5(6):866–874CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Illingworth RS, Bird AP (2009) CpG islands – ‘a rough guide’. FEBS Lett 583(11):1713–1720CrossRefPubMedGoogle Scholar
  11. 11.
    Park JG, Lee SK, Hong IG et al (1994) MDR1 gene expression: its effect on drug resistance to doxorubicin in human hepatocellular carcinoma cell lines. J Natl Cancer Inst 86(9):700–705CrossRefPubMedGoogle Scholar
  12. 12.
    Baker E, El-Osta A (2009) Epigenetics regulation of multidrug resistance 1 gene expression: profiling CpG methylation status using Bisulphite sequencing. In: Zhou J (ed) Multi-drug resistance in cancer. Humana Press, Totowa, NJGoogle Scholar
  13. 13.
    Van Sluis M, McStay B (2015) A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes Dev 29(1):1151–1163CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Stewart-Ornstein J, Lahav G (2016) Dynamics of CDKN1A in single cells defined by an endogenous fluorescent tagging toolkit. Cell Rep 14(7):1800–1811CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Life SciencesImperial College LondonLondonUK

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