Editing DNA Methylation Using CRISPR/Cas9

Gene expression is controlled on several levels from DNA sequence to post-transcriptional changes. Epigenetics demonstrate that gene expression can be controlled by chemical changes in the DNA such as methylation. Since the discovery of epigenetics, researchers have been attempting to produce effective methods to alter the methylation status of select genes. This would enable researchers to effectively turn on or off target genes without affecting the genetic code. Liu et al. (2016) have repurposed the CRISPR/Cas9 system to edit DNA methylation by fusing dCas9 with Tet1 and Dnmt3a, enzymes involved in the methylation/demethylation pathway.

In order to target specific loci, co-expression of sequence specific guide RNAs (gRNAs) were used to guide the dCas9-Tet1 and dCas9-Dnmt3a to the intended locus. The dCas9-Tet1 method was used to target Snrpn-GFP reporter that had been inserted into the Dazl promoter. In ESC Dazl is hypermetylated and therefore not active. However, 3 days post-infected with dCas9-Tet1 some of the cells began to express GFP, indicating successful demethylation. In order to confirm these results, bisulfite-sequencing of the cells’ gDNA was performed and indicated demethylation only occurred in the Snrpn promoter. Similarly dCas9-Dnmt3a was used to target cells and methylate Snrpn-GFP reporter in the GAPDH promoter. By methylating the Snrpn promoter GFP was inactivated within the cells.

Even though there are other editing systems, such a TALEN-based method, Liu et al. (2016) method has been shown to have higher specificity, efficacy and resolution. Researchers compared both systems by determining how well these systems could edit two specific loci, p16 and RHOXF2. When targeting the p16 loci the dCas9-Dnmt3a increased methylation by 25%, while TALE-Dnmt3a only caused a 13% increase. dCas9-Tet1 decreased methylation by 28% in the RHOXF2 promoter region, compared to TALE-Tet1 which caused a 14% decrease in methylation. This effectively shows that dCas9 system is an improvement over the TALEN-based method, due to its specificity.

Due to the successful results of the initial trials, Liu et al. (2016) tested whether their method could be used to change expression levels of certain genes. The dCas9-Tet1 was used to methylate BDNF promoter IV, which is a gene involved in the growth and differentiation of new neurons and synapses. After the system was applied, the BDNF promoter was demethylated and BDNF increased 2-3 folds in post-mitotic neurons. Bisulfite sequencing was later used and confirmed a significant reduction in methylation within this region. They also tested whether fibroblast cells could be reprogrammed into muscle cells by using dCas9-Tet1 with gRNAs to target DMR-5, a methylated distal enhancer in MyoD, and induced MyoD expression, a master regulator for muscle development. There was an increase in MyoD expression levels but it was not sufficient to induce myotube formation. However, when this method was used with the presence of 5-Aza treatment there was a significant increase in myoblast conversion and myotube formation. Nonetheless, these results indicate that this method can effectively turn on or off certain target genes and may even be used to change a cell’s fate.

Liu et al. (2016) method will be useful in order to understand the functional significance of DNA methylation in many biological processes such as gene expression, cell-fate determination, and organization of high order chromatin structure. DNA methylations have been implicated in various disorders such as, atherosclerosis, lupus, muscular dystrophy and cancer. Therefore, the result of this study will be invaluable for treating, understanding and preventing these disorders as it provides not only an insight but a method to edit these methylation sites.

Source: Liu et al., Editing DNA methylation in the Mammalian Genome. Cell, 2016. 167, p 233-247.