Table of Contents

  1. Protocols
  2. Resources
  3. References

Welcome! This resource page provides the information and resources needed to implement a CRISPR/Cas9-targeted DNA methyltransferase system for site-specific induction of DNA methylation. This project is a joint collaboration between the Edwards and Challen labs.

We have used the protocols and resources below to induce methylation and decrease gene expression as described in Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

If you use the protocols on this site, please cite our publication: 

McDonald, J.I., Celik, H., Rois, L.E., Fishberger, G., Fowler, T., Rees, R., Kramer, A., Martens, A., Edwards, J.R., and Challen, G.A. (2016). Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biology Open bio.019067. doi: 10.1242/bio.019067.

Detailed Protocols

  1. Reagent Design:
    1. Design sgRNA to the target region.
    2. Clone the sgRNA.
    3. Order the Cas9 DNA methyltransferase fusions from Addgene. For transient transfection: pCMV-dCas9-D3A (#78256) and pCMV-dCas9-mD3A (#78257). For viral transduction: TETO-dCas9-D3A (#78254) and TETO-dCas9-mD3A (#78255).
  2. Induce DNA Methylation:
    1. Transfection – This protocol is used on HEK293T cells; optimization is likely needed for other cell lines.
    2. Transduction – This protocol is used on mouse 32D cells; optimization is likely needed for other cell lines.
  3. Methylation Analysis:
    1. Amplicon bisulfite sequencing – create Illumina sequencing libraries from the PCR amplicons of your target region after bisulfite conversion.
    2. Sanger bisulfite sequencing – sequence clones of the PCR amplicon of your target region after bisulfite conversion into a sequencing plasmid and sequence it.

If you have any questions please email us at: jredwards<at>wustl.edu 

Resources

Links to groups/organizations/companies whose services we used in the above protocols:

  1. Addgene plasmid depository
  2. Center for Genome Sciences and Systems Biology Spike-In Cooperative for information on spike-in sequencing
  3. GeneWiz Sanger sequencing service
  4. IDT oligonucleotide synthesis service
  5. Zymo Research Bisulfite Primer Seeker online bisulfite primer design tool

Links to the reagents, enzymes, and kits used in the above protocols are included below (roughly in order encountered in the protocols):

  1. MLM3636 sgRNA expression plasmid (Addgene #43860)
  2. BsmBI restriction enzyme
  3. NEB Quick Ligase
  4. NEB 10β competent cells
  5. Lipofectamine LTX transfection reagent
  6. Zymo Research Quick gDNA Miniprep gDNA extraction kit
  7. Zymo Research Quick RNA Miniprep RNA extraction kit
  8. Zymo Research EZ DNA Methylation bisulfite conversion kit
  9. Qiagen PyroMark kit for amplification of bisulfite converted DNA
  10. SPRI beads (aka Agencourt AMPure XP beads)
  11. Qiagen QiaQuick DNA purification columns -- PCR purification and gel extraction
  12. Qiagen Minelute DNA purification columns -- PCR purification and gel extraction
  13. NEB Phusion High Fidelity DNA polymerase
  14. Promega pGEM-T Easy TA cloning vector system
  15. Qubit DNA/RNA quantification assays

References

Here is an aging but good review of epigenetic editing: Groote, M. L. de, Verschure, P. J. & Rots, M. G. Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes. Nucl. Acids Res. 40, 10596–10613 (2012).

The citation information for previous induced methylation publications is below:

  1. Vojta, A. et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucl. Acids Res. gkw159 (2016). doi:10.1093/nar/gkw159.
  2. Cui, C. et al. P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis. Genome Biology 16, 252 (2015).
  3. Bernstein, D. L., Le Lay, J. E., Ruano, E. G. & Kaestner, K. H. TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts. Journal of Clinical Investigation 125, 1998–2006 (2015).
  4. Siddique, A. N. et al. Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a–Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity. Journal of Molecular Biology 425, 479–491 (2013).
  5. Rivenbark, A. G. et al. Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics 7, 350–360 (2012).
  6. van der Gun, B. T. F. et al. Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule. Bioconjugate Chem. 21, 1239–1245 (2010).
  7. Smith, A. E., Hurd, P. J., Bannister, A. J., Kouzarides, T. & Ford, K. G. Heritable Gene Repression through the Action of a Directed DNA Methyltransferase at a Chromosomal Locus. J. Biol. Chem. 283, 9878–9885 (2008).
  8. Li, F. et al. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes. Nucl. Acids Res. 35, 100–112 (2007).
  9. Nomura, W. & Barbas, C. F. In Vivo Site-Specific DNA Methylation with a Designed Sequence-Enabled DNA Methylase. J. Am. Chem. Soc. 129, 8676–8677 (2007).
  10. Smith, A. E. & Ford, K. G. Specific targeting of cytosine methylation to DNA sequences in vivo. Nucleic Acids Res 35, 740–754 (2007).
  11. Li, H. et al. The Histone Methyltransferase SETDB1 and the DNA Methyltransferase DNMT3A Interact Directly and Localize to Promoters Silenced in Cancer Cells. J. Biol. Chem. 281, 19489–19500 (2006).
  12. Minczuk, M., Papworth, M. A., Kolasinska, P., Murphy, M. P. & Klug, A. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc Natl Acad Sci U S A 103, 19689–19694 (2006).
  13. Carvin, C. D., Parr, R. D. & Kladde, M. P. Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins. Nucleic Acids Res 31, 6493–6501 (2003).
  14. McNamara, A. R., Hurd, P. J., Smith, A. E. F. & Ford, K. G. Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships. Nucleic Acids Res 30, 3818–3830 (2002).
  15. Xu, G.-L. & Bestor, T. H. Cytosine methylation targetted to pre-determined sequences. Nat Genet 17, 376–378 (1997).