Editing Methylation States

Editing DNA nucleotide sequences has become common place but using the programmability of RNA-guided endonucleases to affect other changes to the genome are less developed although their potential for making such changes is quite high.  Liu et al. report on their successful efforts to develop a programmable epigenetic writer and eraser system for mammalian systems.

Disabled Cas9 (dCas9) has no DNA endonuclease activity but retains all of the protein’s other DNA interaction characteristics when associated with a guideRNA (gRNA).  Consequently dCas9 can serve as a programmable DNA-binding platform to which other functions can be attached.

DNA methylation by DNMT. Image Credit: http://pubs.niaaa.nih.gov/publications/arcr351/6-16.htm

DNA methylation by DNMT. Image Credit: http://pubs.niaaa.nih.gov/publications/arcr351/6-16.htm

Liu et al. attached  a “CpG-demethylase” and a “CpG-methylase” to dCas9 and programmed it to act on sequences found in  promoters whose activity are sensitive to their methylation state.

The “CpG-demethylase” is the catalytic domain of TET1, a ten-eleven-translocation dioxygenase.  TET1 oxidizes the methyl group on a methylcytosine to form hydroxymethyl cytosine which eventually leads to restoration of a demethylated cytosine following base excision repair of the hydroxmethylated base.

The “CpG-methylase” is the DNA methyltransferase DNMT3a.

dCas9 lacks endonuclease activity. Image Credit: http://www.tolobio.com

dCas9 lacks endonuclease activity. Image Credit: http://www.tolobio.com

Liu et al. tested the efficacy of their system in a mouse embryonic cell line with a GFP reporter under the control of a promoter whose methylation state determines its activity – the Snrpn promoter.  Because the SnrpnGFP transgene is sitting in at a locus thata is hypermethylated, it is not expressed.  After introducing dCas9-TET1 and appropriate gRNAs using lentiviruses Liu et al. found a significant increase in the number of GFP-expressing cells relative to control cells.

To test the efficacy of their programmable methyltransferase system they also used a cell line with a SnrpnGFP reporter at a locus that is usually not-methylated.  Again, after introducing dCas9-Dnmt3a and appropriate gRNAs using lentiviruses Liu et al. found that a significant decrease in the number of GFP-expressing cells relative to control cells.

With functionality of their programmable C-methyl-writing and -erasing system established the authors tested it on endogenous loci in different cell lines that are known to be methylation sensitive and in all cases there was good evidence that the effectors worked as programmed.

Finally, the Liu et al. tested their system in vivo using a transgenic line of mice that carries a transgene whose expression is methylation sensitive.  These experiments were performed using local lentivirus infections of different tissues and further demonstrated the efficacy of the read/write system of Liu et al.

It should be noted that in addition to the current report by Liu et al. there have been other reports recently that are very similar – Vojta et al 2016; Choudhury et al 2016; Xu et al. 2016).

Although all of these reports use mammalian model systems they either may be portable to insects or can be used as template from which insect programmable epigenetic read/write systems can be constructed.

Liu, X. Shawn et al. (2016) Editing DNA Methylation in the Mammalian Genome, Cell , Volume 167 , Issue 1 , 233 – 247.e17

Vojta, A., Dobrinić, P., Tadić, V., Bočkor, L., Korać, P., Julg, B., Klasić, M., and Zoldoš, V.  (2016)  Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44: 5615–5628

Xu, X., Tao, Y., Gao, X., Zhang, L., Li, X., Zou, W., Ruan, K., Wang, F., Xu, G.L., and Hu, R. (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2: 16009

Choudhury, S.R., Cui, Y., Lubecka, K., Stefanska, B., and Irudayaraj, J. (2016) CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget.  DOI: http://dx.doi.org/10.18632/oncotarget

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